Purine derivatives for use in the treatment of allergic, inflammatory and infectious diseases

ABSTRACT

The present invention relates to compounds of formula (I): 
                         
wherein R 1  is C 1-6 alkylamino, or C 1-6 alkoxy; m is an integer having a value of 3, 4, or 5; n is an integer having a value of 0 to 3; p is an integer having a value of 1 or 2 and salts thereof are inducers of human interferon. Compounds which induce human interferon may be useful in the treatment of various disorders, for example the treatment of allergic diseases and other inflammatory conditions for example allergic rhinitis and asthma, the treatment of infectious diseases and cancer, and may also be useful as vaccine adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed pursuant to 35 U.S.C. §371 as a United StatesNational Phase Application of International Patent Application SerialNo. PCT/EP2009/060262 filed Aug. 7, 2009, which claims priority fromU.S. Provisional Application No. 61/087,785 filed on Aug. 11, 2008.

BACKGROUND OF THE INVENTION

The present invention relates to compounds, processes for theirpreparation, compositions containing them, to their use in the treatmentof various disorders in particular allergic diseases and otherinflammatory conditions for example allergic rhinitis and asthma,infectious diseases, cancer, and as vaccine adjuvants.

Vertebrates are constantly threatened by the invasion of microorganismsand have evolved mechanisms of immune defence to eliminate infectivepathogens. In mammals, this immune system comprises two branches; innateimmunity and acquired immunity. The first line of host defence is theinnate immune system, which is mediated by macrophages and dendriticcells. Acquired immunity involves the elimination of pathogens at thelate stages of infection and also enables the generation ofimmunological memory. Acquired immunity is highly specific, due to thevast repertoire of lymphocytes with antigen-specific receptors that haveundergone gene rearrangement.

The innate immune response was originally thought to be non-specific,but is now known to be able to discriminate between self and a varietyof pathogens. The innate immune system recognises microbes via a limitednumber of germline-encoded Pattern-Recognition Receptors (PRRs) whichhave a number of important characteristics.

Toll-like receptors (TLRs) are a family of ten Pattern RecognitionReceptors described in man. TLRs are expressed predominantly by innateimmune cells where their role is to monitor the environment for signs ofinfection and, on activation, mobilise defence mechanisms aimed at theelimination of invading pathogens. The early innate immune-responsestriggered by TLRs limit the spread of infection, while thepro-inflammatory cytokines and chemokines that they induce lead torecruitment and activation of antigen presenting cells, B cells, and Tcells. The TLRs can modulate the nature of the adaptive immune-responsesto give appropriate protection via dendritic cell-activation andcytokine release (Akira S. et al, Nat. Immunol., 2001: 2, 675-680). Theprofile of the response seen from different TLR agonists depends on thecell type activated.

TLR7 is a member of the subgroup of TLRs (TLRs 3, 7, 8, and 9),localised in the endosomal compartment of cells which have becomespecialised to detect non-self nucleic acids. TLR7 plays a key role inanti-viral defence via the recognition of ssRNA (Diebold S. S. et al,Science, 2004: 303, 1529-1531; and Lund J. M. et al, PNAS, 2004: 101,5598-5603). TLR7 has a restricted expression-profile in man and isexpressed predominantly by B cells and plasmacytoid dendritic cells(pDC), and to a lesser extent by monocytes. Plasmacytoid DCs are aunique population of lymphoid-derived dendritic cells (0.2-0.8% ofPeripheral Blood Mononuclear Cells (PBMCs)) which are the primary typeinterferon-producing cells secreting high levels of interferon-alpha(IFNα) and interferon-beta (IFNβ) in response to viral infections (LiuY-J, Annu. Rev. Immunol., 2005: 23, 275-306).

Allergic diseases are associated with a Th2-biased immune-response toallergens. Th2 responses are associated with raised levels of IgE,which, via its effects on mast cells, promotes a hypersensitivity toallergens, resulting in the symptoms seen, for example, in allergicrhinitis. In healthy individuals the immune-response to allergens ismore balanced with a mixed Th2/Th1 and regulatory T cell response. TLR7ligands have been shown to reduce Th2 cytokine and enhance Th1 cytokinerelease in vitro and to ameliorate Th2-type inflammatory responses inallergic lung models in vivo (Fili L. et al, J. All. Clin. Immunol.,2006: 118, 511-517; Moisan J. et al, Am. J. Physiol. Lung Cell Mol.Physiol., 2006: 290, L987-995; Tao et al, Chin. Med. J., 2006: 119,640-648). Thus TLR7 ligands have the potential to rebalance theimmune-response seen in allergic individuals and lead to diseasemodification.

Central to the generation of an effective innate immune response inmammals are mechanisms which bring about the induction of interferonsand other cytokines which act upon cells to induce a number of effects.These effects can include the activation of anti-infective geneexpression, the activation of antigen presentation in cells to drivestrong antigen-specific immunity and the promotion of phagocytosis inphagocytic cells.

Interferon was first described as a substance which could protect cellsfrom viral infection (Isaacs & Lindemann, J. Virus Interference. Proc.R. Soc. Lon. Ser. B. Biol. Sci. 1957: 147, 258-267). In man, the type Iinterferons are a family of related proteins encoded by genes onchromosome 9 and encoding at least 13 isoforms of interferon alpha(IFNα) and one isoform of interferon beta (IFNβ). Recombinant IFNα wasthe first approved biological therapeutic and has become an importanttherapy in viral infections and in cancer. As well as direct antiviralactivity on cells, interferons are known to be potent modulators of theimmune response, acting on cells of the immune system.

As a first-line therapy for hepatitis C virus (HCV) disease, interferoncombinations can be highly effective at reducing viral load and in somesubjects in eliminating viral replication. However, many patients failto show a sustained viral response and in these patients viral load isnot controlled. Additionally, therapy with injected interferon may beassociated with a number of unwanted adverse effects which are shown toaffect compliance (Dudley T, et al, Gut., 2006: 55(9), 1362-3).

Administration of a small molecule compound which could stimulate theinnate immune response, including the activation of type I interferonsand other cytokines, could become an important strategy for thetreatment or prevention of human diseases including viral infections.This type of immunomodulatory strategy has the potential to identifycompounds which may be useful not only in infectious diseases but alsoin cancer (Krieg. Curr. Oncol. Rep., 2004: 6(2), 88-95), allergicdiseases (Moisan J. et al, Am. J. Physiol. Lung Cell Mol. Physiol.,2006: 290, L987-995), other inflammatory conditions such as irritablebowel disease (Rakoff-Nahoum S., Cell., 2004, 23, 118(2): 229-41), andas vaccine adjuvants (Persing et al. Trends Microbiol. 2002: 10 (10Suppl), S32-7).

In animal models, imiquimod demonstrated adjuvant activities eithertopically (Adams S. et al, J. Immunol., 2008, 181:776-84; Johnston D. etal, Vaccine, 2006, 24:1958-65), or systemically (Fransen F. et al,Infect. Immun., 2007, 75:5939-46). Resiquimod and other related TLR7/8agonists have also been shown to display adjuvant activity (Ma R. et al,Biochem. Biophys. Res. Commun., 2007, 361:537-42; Wille-Reece U. et al,Proc. Natl. Acad. Sci. USA, 2005, 102:15190-4; Wille-Reece U. et al,US2006045885 A1).

Mechanisms which lead to induction of type I interferons are only partlyunderstood. One mechanism which can lead to the induction of interferonin many cell types is the recognition of double-stranded viral RNA bythe RNA helicases RIG-I and MDA5. This mechanism is thought to be theprimary mechanism by which interferons are induced by Sendai virusinfection of cells.

Further mechanisms for the induction of interferons are viaTLR-dependent signalling events. In man, plasmacytoid dendritic cells(pDCs) are professional interferon-producing cells, able to make largeamounts of interferons in response to, for example, viral infection.These pDCs are shown to preferentially express TLR7 and TLR9 andstimulation of these receptors with viral RNA or DNA respectively caninduce expression of interferon alpha.

Oligonucleotide agonists of TLR7 and TLR9, and small moleculepurine-based agonists of TLR7 have been described which can induceinterferon alpha from these cell types in animals and in man (Takeda K.et al, Annu. Rev. Immunol., 2003: 21, 335-76). TLR7 agonists includeimidazoquinoline compounds such as imiquimod and resiquimod, oxoadenineanalogues and also nucleoside analogues such as loxoribine and7-thia-8-oxoguanosine which have long been known to induce interferonalpha.

It remains unclear how small molecule purine-like compounds can inducetype I interferons and other cytokines since the molecular targets ofthese known inducers have not been identified. However, an assaystrategy has been developed to characterise small molecule inducers ofhuman interferon IFNα (regardless of mechanism) which is based onstimulation of primary human donor cells with compounds, and isdisclosed herein.

BRIEF DESCRIPTION OF THE INVENTION

Certain compounds of the invention have been shown to be inducers ofhuman interferon and may possess an improved profile with respect toknown inducers of human interferon, for example enhanced potency, andmay show enhanced selectivity for IFNα with respect to TNFα. Forexample, certain compounds of the invention indicate greater than100-fold selectivity for IFNα induction over TNFα induction. Compoundswhich induce human interferon may be useful in the treatment of variousdisorders, for example the treatment of allergic diseases and otherinflammatory conditions for example allergic rhinitis and asthma, thetreatment of infectious diseases and cancer, and may also be useful asvaccine adjuvants.

Certain compounds of the invention are potent immunomodulators andaccordingly, care should be exercised in their handling.

SUMMARY OF THE INVENTION

In a first aspect, there are provided compounds of formula (I):

wherein;

-   -   R¹ is C₁₋₆alkylamino, or C₁₋₆alkoxy;    -   m is an integer having a value of 3, 4, or 5;    -   n is an integer having a value of 0 to 3;    -   p is an integer having a value of 1 or 2;        and salts thereof.

In a further embodiment, R¹ is n-butyloxy.

In a further embodiment, R¹ is (1S)-1-methylbutyloxy.

In a further embodiment, R¹ is n-butylamino.

In a further embodiment, m is 3.

In a further embodiment, m is 4.

In a further embodiment, m is 5.

In a further embodiment, n is 0.

In a further embodiment, n is 1.

In a further embodiment, n is 2.

In a further embodiment, when n is 0, the stereochemistry at the pointof attachment of the —(CH₂)_(m)—NH— group to the

group is in the R-configuration.

In a further embodiment, when n is 0, the stereochemistry at the pointof attachment of the —(CH₂)_(m)—NH— group to the

group is in the S-configuration.

In a further embodiment, p is 1.

In a further embodiment, p is 2.

There exists a subset of compounds of formula (I), being of formula(I′). Accordingly, in a further aspect, there are provided compounds offormula (I′):

wherein;

-   -   R^(1′) is C₁₋₆alkylamino, or C₁₋₆alkoxy;    -   m′ is an integer having a value of 3;    -   n′ is an integer having a value of 0 to 3;    -   p′ is an integer having a value of 1 or 2;        and salts thereof.

In a further embodiment, R^(1′) is n-butoxy.

In a further embodiment, R^(1′) is (1S)-1-methylbutyl]oxy.

In a further embodiment, R^(1′) is n-butylamino.

In a further embodiment, n′ is 0.

In a further embodiment, n′ is 1.

In a further embodiment, n′ is 2.

In a further embodiment, p′ is 1.

In a further embodiment, p′ is 2.

Examples of compounds of formula (I) are provided in the following list,and form a further aspect of the invention:

-   6-amino-2-(butyloxy)-9-{3-[(2R)-tetrahydro-2-furanylamino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-{3-[(2S)-tetrahydro-2-furanylamino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-{3-[(tetrahydro-3-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-{3-[(tetrahydro-2H-pyran-2-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-{3-[(tetrahydro-2H-pyran-3-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-2-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-3-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-4-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-(3-{[2-(tetrahydro-2H-pyran-2-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[4-(tetrahydro-2H-pyran-4-ylamino)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(tetrahydro-2H-pyran-4-ylamino)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]butyl}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{5-[(tetrahydro-2H-pyran-4-ylmethyl)amino]pentyl}-7,9-dihydro-8H-purin-8-one-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(3R)-tetrahydro-3-furanylamino]butyl}-7,9-dihydro-8H-purin-8-one,    and;-   6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(3S)-tetrahydro-3-furanylamino]butyl}-7,9-dihydro-8H-purin-8-one;    and salts thereof.

There is thus provided as a further aspect of the invention a compoundof formula (I), or a pharmaceutically acceptable salt thereof, for useas in therapy.

It will be appreciated that, when a compound of formula (I) or apharmaceutically acceptable salt thereof is used in therapy, it is usedas an active therapeutic agent.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofallergic diseases and other inflammatory conditions, infectiousdiseases, and cancer.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofallergic rhinitis.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofasthma.

There is also therefore provided a vaccine adjuvant comprising compoundof formula (I), or a pharmaceutically acceptable salt thereof.

There is further provided an immugenic composition comprising an antigenor antigen composition and a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a vaccine composition comprising an antigen orantigen composition and a compound of formula (I), or a pharmaceuticallyacceptable salt thereof.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, an immugenic composition comprising an antigenor antigen composition and a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treating or preventing diseasecomprising the administration to a patient human subject suffering fromor susceptible to disease, a vaccine composition comprising an antigenor antigen composition and a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of animmugenic composition comprising an antigen or antigen composition, forthe treatment or prevention of disease.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of avaccine composition comprising an antigen or antigen composition, forthe treatment or prevention of disease.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of allergic diseases and other inflammatoryconditions, infectious diseases, and cancer.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of allergic rhinitis.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of asthma.

There is further provided a method of treatment of allergic rhinitis,which method comprises administering to a human subject in need thereofa therapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treatment of asthma, which methodcomprises administering to a human subject in need thereof atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

The invention provides in a further aspect, a combination comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,together with at least one other therapeutically active agent.

There is further provided a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,and one or more pharmaceutically acceptable diluents or carriers.

There is also provided a process for preparing a pharmaceuticalcomposition which comprises admixing a compound of formula (I), or apharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable diluents or carriers.

The compounds of formula (I) and salts thereof may be prepared by themethodology described herein, which constitutes a further aspect of thisinvention.

Accordingly, there is provided a process for the preparation of acompound of formula (I), which process comprises the deprotection of acompound of formula (II):

wherein R¹, m, n, and p are as hereinbefore defined for a compound offormula (I) and R² is C₁₋₆alkyl, and thereafter, if required, carryingout one or more of the following optional steps:(i). removing any necessary protecting group;(ii). preparing a salt of the compound so-formed.

The present invention covers all combinations of embodiments and aspectsherein described.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is described in terms known and appreciated bythose skilled in the art. For ease of reference certain termshereinafter are defined. The fact that certain terms are defined,however, should not be considered as indicative that defined terms areused in a manner inconsistent with the ordinary meaning or,alternatively, that any term that is undefined is indefinite or not usedwithin the ordinary and accepted meaning. Rather, all terms used hereinare believed to describe the invention such that one of ordinary skillcan appreciate the scope of the present invention. The followingdefinitions are meant to clarify, but not limit, the terms defined.

References to ‘alkyl’ include references to both straight-chain andbranched-chain aliphatic isomers of the corresponding alkyl containingup to six carbon atoms, for example up to four carbon atoms or up to twocarbon atoms. Such references to ‘alkyl’ are also applicable when analkyl group is part of another group, for example an alkylamino oralkoxy group. Examples of such alkyl groups and groups containing alkylgroups are C₁₋₆alkyl, C₁₋₆alkylamino, and C₁₋₆alkoxy.

References to ‘heterocycle’ or ‘heterocyclyl’ refer to a monocyclicsaturated heterocyclic aliphatic ring containing 3-6 carbon atoms andone heteroatom, which heteroatom is nitrogen. Such heterocyclic ring areazetidine or azetidinyl, pyrrolidine or pyrrolidinyl, piperidine orpiperidinyl, and azepine or azepinyl.

References to ‘halogen’ refer to iodine, bromine, chlorine or fluorine,typically fluorine, bromine, or chlorine. References to ‘halo’ refer toiodo, bromo, chloro or fluoro, typically fluoro, bromo, or chloro.

It is to be understood that references herein to compounds of theinvention mean a compound of formula (I) as the free base, or as a saltfor example a pharmaceutically acceptable salt.

Salts of the compounds of formula (I) include pharmaceuticallyacceptable salts and salts which may not be pharmaceutically acceptablebut may be useful in the preparation of compounds of formula (I) andpharmaceutically acceptable salts thereof. Salts may be derived fromcertain inorganic or organic acids, or certain inorganic or organicbases.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the salts of the compounds of formula (I).

Examples of salts are pharmaceutically acceptable salts.Pharmaceutically acceptable salts include acid addition salts and baseaddition salts. For a review on suitable salts see Berge et al., J.Pharm. Sci., 66:1-19 (1977).

Examples of pharmaceutically acceptable acid addition salts of acompound of formula (I) include hydrobromide, hydrochloride, sulphate,p-toluenesulphonate, methanesulphonate, naphthalenesulphonate, andphenylsulphonate salts.

Examples of pharmaceutically acceptable base salts include alkali metalsalts such as those of sodium and potassium, and alkaline earth metalsalts such as those of calcium and magnesium.

Salts may be formed using techniques well-known in the art, for exampleby precipitation from solution followed by filtration, or by evaporationof the solvent.

Typically, a pharmaceutically acceptable acid addition salt can beformed by reaction of a compound of formula (I) with a suitable strongacid (such as hydrobromic, hydrochloric, sulphuric, p-toluenesulphonic,methanesulphonic or naphthalenesulphonic acids), optionally in asuitable solvent such as an organic solvent, to give the salt which isusually isolated for example by crystallisation and filtration.

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallised. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or solvents with a high propensity to formhydrogen bonds such as water, ethanol, iso-propyl alcohol, andN-methylpyrrolidinone may be used to form solvates. Methods for theidentification of solvated include, but are not limited to, NMR andmicroanalysis. Solvates of the compounds of formula (I) are within thescope of the invention. As used herein, the term solvate encompassessolvates of both a free base compound as well as any salt thereof.

Certain of the compounds of the invention may contain chiral atomsand/or multiple bonds, and hence may exist in one or more stereoisomericforms. The present invention encompasses all of the stereoisomers of thecompounds of the invention, including optical isomers, whether asindividual stereoisomers or as mixtures thereof including racemicmodifications. Any stereoisomer may contain less than 10% by weight, forexample less than 5% by weight, or less than 0.5% by weight, of anyother stereoisomer. For example, any optical isomer may contain lessthan 10% by weight, for example less than 5% by weight, or less than0.5% by weight, of its antipode.

Certain of the compounds of the invention may exist in tautomeric forms.It will be understood that the present invention encompasses all of thetautomers of the compounds of the invention whether as individualtautomers or as mixtures thereof.

The compounds of the invention may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compounds of theinvention may exist as polymorphs, all of which are included within thescope of the present invention. The most thermodynamically stablepolymorphic form or forms of the compounds of the invention are ofparticular interest.

Polymorphic forms of compounds of the invention may be characterised anddifferentiated using a number of conventional analytical techniques,including, but not limited to, X-ray powder diffraction (XRPD), infraredspectroscopy (IR), Raman spectroscopy, differential scanning calorimetry(DSC), thermogravimetric analysis (TGA) and solid-state nuclear magneticresonance (ssNMR).

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, hydrates, isomers and polymorphic formsof the compounds of formula (I) and salts and solvates thereof.

Examples of disease states in which the compounds of formula (I) andpharmaceutically acceptable salts thereof have potentially beneficialeffects include allergic diseases and other inflammatory conditions forexample allergic rhinitis and asthma, infectious diseases, and cancer.The compounds of formula (I) and pharmaceutically acceptable saltsthereof are also of potential use as vaccine adjuvants.

As modulators of the immune response the compounds of formula (I) andpharmaceutically acceptable salts thereof may also be useful, asstand-alone or in combination as an adjuvant, in the treatment and/orprevention of immune-mediated disorders, including but not limited toinflammatory or allergic diseases such as asthma, allergic rhinitis andrhinoconjuctivitis, food allergy, hypersensitivity lung diseases,eosinophilic pneumonitis, delayed-type hypersensitivity disorders,atherosclerosis, pancreatitis, gastritis, colitis, osteoarthritis,psoriasis, sarcoidosis, pulmonary fibrosis, respiratory distresssyndrome, bronchiolitis, chronic obstructive pulmonary disease,sinusitis, cystic fibrosis, actinic keratosis, skin dysplasia, chronicurticaria, eczema and all types of dermatitis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment and/or prevention ofreactions against respiratory infections, including but not limited toairways viral exacerbations and tonsillitis. The compounds may also beuseful in the treatment and/or prevention of autoimmune diseasesincluding but not limited to rheumatoid arthritis, psoriatic arthritis,systemic lupus erythematosus, Sjöegrens disease, ankylosing spondylitis,scleroderma, dermatomyositis, diabetes, graft rejection, includinggraft-versus-host disease, inflammatory bowel diseases including, butnot limited to, Crohn's disease and ulcerative colitis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment of infectious diseasesincluding, but not limited to, those caused by hepatitis viruses (e.g.hepatitis B virus, hepatitis C virus), human immunodeficiency virus,papillomaviruses, herpesviruses, respiratory viruses (e.g. influenzaviruses, respiratory syncytial virus, rhinovirus, metapneumovirus,parainfluenzavirus, SARS), and West Nile virus. The compounds of formula(I) and pharmaceutically acceptable salts thereof may also be useful inthe treatment of microbial infections caused by, for example, bacteria,fungi, or protozoa. These include, but are not limited to, tuberculosis,bacterial pneumonia, aspergillosis, histoplasmosis, candidosis,pneumocystosis, leprosy, chlamydia, cryptococcal disease,cryptosporidosis, toxoplasmosis, leishmania, malaria, andtrypanosomiasis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment of various cancers, inparticular the treatment of cancers that are known to be responsive toimmunotherapy and including, but not limited to, renal cell carcinoma,lung cancer, breast cancer, colorectal cancer, bladder cancer, melanoma,leukaemia, lymphomas and ovarian cancer.

It will be appreciated by those skilled in the art that referencesherein to treatment or therapy may, depending on the condition, extendto prophylaxis as well as the treatment of established conditions.

As mentioned herein, compounds of formula (I) and pharmaceuticallyacceptable salts thereof may be useful as therapeutic agents.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be formulated for administration in any convenient way.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may, for example, be formulated for oral, topical, inhaled,intranasal, buccal, parenteral (for example intravenous, subcutaneous,intradermal, or intramuscular) or rectal administration. In one aspect,the compounds of formula (I) and pharmaceutically acceptable saltsthereof are formulated for oral administration. In a further aspect, thecompounds of formula (I) and pharmaceutically acceptable salts thereofare formulated for topical administration, for example intranasal orinhaled administration.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, mucilage of starch, cellulose or polyvinylpyrrolidone; fillers, for example, lactose, microcrystalline cellulose,sugar, maize starch, calcium phosphate or sorbitol; lubricants, forexample, magnesium stearate, stearic acid, talc, polyethylene glycol orsilica; disintegrants, for example, potato starch, croscarmellose sodiumor sodium starch glycollate; or wetting agents such as sodium laurylsulphate. The tablets may be coated according to methods well known inthe art.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may contain conventionaladditives such as suspending agents, for example, sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats; emulsifying agents, for example, lecithin, sorbitan mono-oleate oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters, propyleneglycol or ethyl alcohol; or preservatives, for example, methyl or propylp-hydroxybenzoates or sorbic acid. The preparations may also containbuffer salts, flavouring, colouring and/or sweetening agents (e.g.mannitol) as appropriate.

Compositions for intranasal administration include aqueous compositionsadministered to the nose by drops or by pressurised pump. Suitablecompositions contain water as the diluent or carrier for this purpose.Compositions for administration to the lung or nose may contain one ormore excipients, for example one or more suspending agents, one or morepreservatives, one or more surfactants, one or more tonicity adjustingagents, one or more co-solvents, and may include components to controlthe pH of the composition, for example a buffer system. Further, thecompositions may contain other excipients such as antioxidants, forexample sodium metabisulphite, and taste-masking agents. Compositionsmay also be administered to the nose or other regions of the respiratorytract by nebulisation.

Intranasal compositions may permit the compound(s) of formula (I) or (a)pharmaceutically acceptable salt(s) thereof to be delivered to all areasof the nasal cavities (the target tissue) and further, may permit thecompound(s) of formula (I) or (a) pharmaceutically acceptable salt(s)thereof to remain in contact with the target tissue for longer periodsof time. A suitable dosing regime for intranasal compositions would befor the patient to inhale slowly through the nose subsequent to thenasal cavity being cleared. During inhalation the composition would beadministered to one nostril while the other is manually compressed. Thisprocedure would then be repeated for the other nostril. Typically, oneor two sprays per nostril would be administered by the above procedureone, two, or three times each day, ideally once daily. Of particularinterest are intranasal compositions suitable for once-dailyadministration.

The suspending agent(s), if included, will typically be present in anamount of from 0.1 to 5% (w/w), such as from 1.5% to 2.4% (w/w), basedon the total weight of the composition. Examples of pharmaceuticallyacceptable suspending agents include, but are not limited to, Avicel®(microcrystalline cellulose and carboxymethylcellulose sodium),carboxymethylcellulose sodium, veegum, tragacanth, bentonite,methylcellulose, xanthan gum, carbopol and polyethylene glycols.

Compositions for administration to the lung or nose may contain one ormore excipients may be protected from microbial or fungal contaminationand growth by inclusion of one or more preservatives. Examples ofpharmaceutically acceptable anti-microbial agents or preservativesinclude, but are not limited to, quaternary ammonium compounds (forexample benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, lauralkonium chloride and myristyl picoliniumchloride), mercurial agents (for example phenylmercuric nitrate,phenylmercuric acetate and thimerosal), alcoholic agents (for examplechlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterialesters (for example esters of para-hydroxybenzoic acid), chelatingagents such as disodium edetate (EDTA) and other anti-microbial agentssuch as chlorhexidine, chlorocresol, sorbic acid and its salts (such aspotassium sorbate) and polymyxin. Examples of pharmaceuticallyacceptable anti-fungal agents or preservatives include, but are notlimited to, sodium benzoate, sorbic acid, sodium propionate,methylparaben, ethylparaben, propylparaben and butylparaben. Thepreservative(s), if included, may be present in an amount of from 0.001to 1% (w/w), such as from 0.015% to 0.5% (w/w) based on the total weightof the composition.

Compositions (for example wherein at least one compound is insuspension) may include one or more surfactants which functions tofacilitate dissolution of the medicament particles in the aqueous phaseof the composition. For example, the amount of surfactant used is anamount which will not cause foaming during mixing. Examples ofpharmaceutically acceptable surfactants include fatty alcohols, estersand ethers, such as polyoxyethylene (20) sorbitan monooleate(Polysorbate 80), macrogol ethers, and poloxamers. The surfactant may bepresent in an amount of between about 0.01 to 10% (w/w), such as from0.01 to 0.75% (w/w), for example about 0.5% (w/w), based on the totalweight of the composition.

One or more tonicity adjusting agent(s) may be included to achievetonicity with body fluids e.g. fluids of the nasal cavity, resulting inreduced levels of irritancy. Examples of pharmaceutically acceptabletonicity adjusting agents include, but are not limited to, sodiumchloride, dextrose, xylitol, calcium chloride, glucose, glycerine andsorbitol. A tonicity-adjusting agent, if present, may be included in anamount of from 0.1 to 10% (w/w), such as from 4.5 to 5.5% (w/w), forexample about 5.0% (w/w), based on the total weight of the composition.

The compositions of the invention may be buffered by the addition ofsuitable buffering agents such as sodium citrate, citric acid,trometamol, phosphates such as disodium phosphate (for example thedodecahydrate, heptahydrate, dihydrate and anhydrous forms), or sodiumphosphate and mixtures thereof.

A buffering agent, if present, may be included in an amount of from 0.1to 5% (w/w), for example 1 to 3% (w/w) based on the total weight of thecomposition.

Examples of taste-masking agents include sucralose, sucrose, saccharinor a salt thereof, fructose, dextrose, glycerol, corn syrup, aspartame,acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycyrrhizinate,thaumatin, neotame, mannitol, menthol, eucalyptus oil, camphor, anatural flavouring agent, an artificial flavouring agent, andcombinations thereof.

One or more co-solvent(s) may be included to aid solubility of themedicament compound(s) and/or other excipients. Examples ofpharmaceutically acceptable co-solvents include, but are not limited to,propylene glycol, dipropylene glycol, ethylene glycol, glycerol,ethanol, polyethylene glycols (for example PEG300 or PEG400), andmethanol. In one embodiment, the co-solvent is propylene glycol.

Co-solvent(s), if present, may be included in an amount of from 0.05 to30% (w/w), such as from 1 to 25% (w/w), for example from 1 to 10% (w/w)based on the total weight of the composition.

Compositions for inhaled administration include aqueous, organic oraqueous/organic mixtures, dry powder or crystalline compositionsadministered to the respiratory tract by pressurised pump or inhaler,for example, reservoir dry powder inhalers, unit-dose dry powderinhalers, pre-metered multi-dose dry powder inhalers, nasal inhalers orpressurised aerosol inhalers, nebulisers or insufflators. Suitablecompositions contain water as the diluent or carrier for this purposeand may be provided with conventional excipients such as bufferingagents, tonicity modifying agents and the like. Aqueous compositions mayalso be administered to the nose and other regions of the respiratorytract by nebulisation. Such compositions may be aqueous solutions orsuspensions or aerosols delivered from pressurised packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.

Compositions for administration topically to the nose (for example, forthe treatment of rhinitis) or to the lung, include pressurised aerosolcompositions and aqueous compositions delivered to the nasal cavities bypressurised pump. Compositions which are non-pressurised and aresuitable for administration topically to the nasal cavity are ofparticular interest. Suitable compositions contain water as the diluentor carrier for this purpose. Aqueous compositions for administration tothe lung or nose may be provided with conventional excipients such asbuffering agents, tonicity-modifying agents and the like. Aqueouscompositions may also be administered to the nose by nebulisation.

A fluid dispenser may typically be used to deliver a fluid compositionto the nasal cavities. The fluid composition may be aqueous ornon-aqueous, but typically aqueous. Such a fluid dispenser may have adispensing nozzle or dispensing orifice through which a metered dose ofthe fluid composition is dispensed upon the application of auser-applied force to a pump mechanism of the fluid dispenser. Suchfluid dispensers are generally provided with a reservoir of multiplemetered doses of the fluid composition, the doses being dispensable uponsequential pump actuations. The dispensing nozzle or orifice may beconfigured for insertion into the nostrils of the user for spraydispensing of the fluid composition into the nasal cavity. A fluiddispenser of the aforementioned type is described and illustrated inInternational Patent Application publication number WO 2005/044354(Glaxo Group Limited). The dispenser has a housing which houses afluid-discharge device having a compression pump mounted on a containerfor containing a fluid composition. The housing has at least onefinger-operable side lever which is movable inwardly with respect to thehousing to move the container upwardly in the housing by means of a camto cause the pump to compress and pump a metered dose of the compositionout of a pump stem through a nasal nozzle of the housing. In oneembodiment, the fluid dispenser is of the general type illustrated inFIGS. 30-40 of WO 2005/044354.

Aqueous compositions containing a compound of formula (I) or apharmaceutically acceptable salt thereof may also be delivered by a pumpas disclosed in International Patent Application publication numberWO2007/138084 (Glaxo Group Limited), for example as disclosed withreference to FIGS. 22-46 thereof, or as disclosed in United Kingdompatent application number GB0723418.0 (Glaxo Group Limited), for exampleas disclosed with reference to FIGS. 7-32 thereof. The pump may beactuated by an actuator as disclosed in FIGS. 1-6 of GB0723418.0.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend compositions generallycontain a powder mix for inhalation of the compound of formula (I) or apharmaceutically acceptable salt thereof and a suitable powder base(carrier/diluent/excipient substance) such as mono-, di-, orpolysaccharides (for example lactose or starch). Dry powder compositionsmay also include, in addition to the drug and carrier, a furtherexcipient (for example a ternary agent such as a sugar ester for examplecellobiose octaacetate, calcium stearate, or magnesium stearate.

In one embodiment, a composition suitable for inhaled administration maybe incorporated into a plurality of sealed dose containers provided onmedicament pack(s) mounted inside a suitable inhalation device. Thecontainers may be rupturable, peelable, or otherwise openableone-at-a-time and the doses of the dry powder composition administeredby inhalation on a mouthpiece of the inhalation device, as known in theart. The medicament pack may take a number of different forms, forinstance a disk-shape or an elongate strip. Representative inhalationdevices are the DISKHALER™ and DISKUS™ devices, marketed byGlaxoSmithKline.

A dry powder inhalable composition may also be provided as a bulkreservoir in an inhalation device, the device then being provided with ametering mechanism for metering a dose of the composition from thereservoir to an inhalation channel where the metered dose is able to beinhaled by a patient inhaling at a mouthpiece of the device. Exemplarymarketed devices of this type are TURBUHALER™ (AstraZeneca), TWISTHALER™(Schering) and CLICKHALER™ (Innovate.)

A further delivery method for a dry powder inhalable composition is formetered doses of the composition to be provided in capsules (one doseper capsule) which are then loaded into an inhalation device, typicallyby the patient on demand. The device has means to rupture, pierce orotherwise open the capsule so that the dose is able to be entrained intothe patient's lung when they inhale at the device mouthpiece. Asmarketed examples of such devices there may be mentioned ROTAHALER™(GlaxoSmithKline) and HANDIHALER™ (Boehringer Ingelheim.)

Pressurised aerosol compositions suitable for inhalation can be either asuspension or a solution and may contain a compound of formula (I) or apharmaceutically acceptable salt thereof and a suitable propellant suchas a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixturesthereof, particularly hydrofluoroalkanes, especially1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or amixture thereof. The aerosol composition may optionally containadditional composition excipients well known in the art such assurfactants e.g. oleic acid, lecithin or an oligolactic acid orderivative thereof e.g. as described in WO 94/21229 and WO 98/34596(Minnesota Mining and Manufacturing Company) and co-solvents e.g.ethanol. Pressurised compositions will generally be retained in acanister (e.g. an aluminium canister) closed with a valve (e.g. ametering valve) and fitted into an actuator provided with a mouthpiece.

Ointments, creams and gels, may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Such bases may thus, for example, includewater and/or an oil such as liquid paraffin or a vegetable oil such asarachis oil or castor oil, or a solvent such as polyethylene glycol.Thickening agents and gelling agents which may be used according to thenature of the base include soft paraffin, aluminium stearate,cetostearyl alcohol, polyethylene glycols, wool-fat, beeswax,carboxypolymethylene and cellulose derivatives, and/or glycerylmonostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will ingeneral also contain one or more emulsifying agents, stabilising agents,dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of anysuitable powder base, for example, talc, lactose or starch. Drops may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing agents, solubilising agents, suspending agents orpreservatives.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may, for example, be formulated for transdermal delivery bycomposition into patches or other devices (e.g. pressurised gas devices)which deliver the active component into the skin.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in the conventional manner.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated as suppositories, e.g. containingconventional suppository bases such as cocoa butter or other glycerides.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated for parenteral administration by bolusinjection or continuous infusion and may be presented in unit dose form,for instance as ampoules, vials, small volume infusions or pre-filledsyringes, or in multidose containers with an added preservative. Thecompositions may take such forms as solutions, suspensions, or emulsionsin aqueous or non-aqueous vehicles, and may contain formulatory agentssuch as anti-oxidants, buffers, antimicrobial agents and/or tonicityadjusting agents. Alternatively, the active ingredient may be in powderform for constitution with a suitable vehicle, e.g. sterile,pyrogen-free water, before use. The dry solid presentation may beprepared by filling a sterile powder aseptically into individual sterilecontainers or by filling a sterile solution aseptically into eachcontainer and freeze-drying.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated with vaccines as adjuvants to modulatetheir activity. Such compositions may contain antibody(ies) or antibodyfragment(s) or an antigenic component including but not limited toprotein, DNA, live or dead bacteria and/or viruses or virus-likeparticles, together with one or more components with adjuvant activityincluding but not limited to aluminium salts, oil and water emulsions,heat shock proteins, lipid A preparations and derivatives, glycolipids,other TLR agonists such as CpG DNA or similar agents, cytokines such asGM-CSF or IL-12 or similar agents.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be employed alone or in combination with other therapeuticagents. The compounds of formula (I) and pharmaceutically acceptablesalts thereof and the other pharmaceutically active agent(s) may beadministered together or separately and, when administered separately,administration may occur simultaneously or sequentially, in any order.The amounts of the compound(s) of formula (I) or (a) pharmaceuticallyacceptable salt(s) thereof and the other pharmaceutically activeagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect. Theadministration of a combination of a compound of formula (I) or apharmaceutically acceptable salt thereof with other treatment agents maybe by administration concomitantly in a unitary pharmaceuticalcomposition including both compounds, or in separate pharmaceuticalcompositions each including one of the compounds. Alternatively, thecombination may be administered separately in a sequential mannerwherein one treatment agent is administered first and the other secondor vice versa. Such sequential administration may be close in time orremote in time.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more agents useful in theprevention or treatment of viral infections. Examples of such agentsinclude, without limitation; polymerase inhibitors such as thosedisclosed in WO 2004/037818-A1, as well as those disclosed in WO2004/037818 and WO 2006/045613; JTK-003, JTK-019, NM-283, HCV-796,R-803, R1728, R1626, as well as those disclosed in WO 2006/018725, WO2004/074270, WO 2003/095441, US2005/0176701, WO 2006/020082, WO2005/080388, WO 2004/064925, WO 2004/065367, WO 2003/007945, WO02/04425, WO 2005/014543, WO 2003/000254, EP 1065213, WO 01/47883, WO2002/057287, WO 2002/057245 and similar agents; replication inhibitorssuch as acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine andsimilar agents; protease inhibitors such as the HIV protease inhibitorssaquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir,brecanavir, atazanavir, tipranavir, palinavir, lasinavir, and the HCVprotease inhibitors BILN2061, VX-950, SCHS03034; and similar agents;nucleoside and nucleotide reverse transcriptase inhibitors such aszidovudine, didanosine, lamivudine, zalcitabine, abacavir, stavidine,adefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine,alovudine, amdoxovir, elvucitabine, and similar agents; non-nucleosidereverse transcriptase inhibitors (including an agent havinganti-oxidation activity such as immunocal, oltipraz etc.) such asnevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz,capravirine, TMC-278, TMC-125, etravirine, and similar agents; entryinhibitors such as enfuvirtide (T-20), T-1249, PRO-542, PRO-140,TNX-355, BMS-806, 5-Helix and similar agents; integrase inhibitors suchas L-870,180 and similar agents; budding inhibitors such as PA-344 andPA-457, and similar agents; chemokine receptor inhibitors such asvicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK-427,857), TAK449, aswell as those disclosed in WO 02/74769, WO 2004/054974, WO 2004/055012,WO 2004/055010, WO 2004/055016, WO 2004/055011, and WO 2004/054581, andsimilar agents; neuraminidase inhibitors such as CS-8958, zanamivir,oseltamivir, peramivir and similar agents; ion channel blockers such asamantadine or rimantadine and similar agents; and interfering RNA andantisense oligonucleotides and such as ISIS-14803 and similar agents;antiviral agents of undetermined mechanism of action, for example thosedisclosed in WO 2005/105761, WO 2003/085375, WO 2006/122011, ribavirin,and similar agents. The compounds of formula (I) and pharmaceuticallyacceptable salts thereof may also be used in combination with one ormore other agents which may be useful in the prevention or treatment ofviral infections for example immune therapies (e.g. interferon or othercytokines/chemokines, cytokine/chemokine receptor modulators, cytokineagonists or antagonists and similar agents); and therapeutic vaccines,antifibrotic agents, anti-inflammatory agents such as corticosteroids ornon-steroidal anti-inflammatory agents (NSAIDs) and similar agents.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more other agents whichmay be useful in the prevention or treatment of allergic disease,inflammatory disease, autoimmune disease, for example; antigenimmunotherapy, anti-histamines, steroids, NSAIDs, bronchodilators (e.g.beta 2 agonists, adrenergic agonists, anticholinergic agents,theophylline), methotrexate, leukotriene modulators and similar agents;monoclonal antibody therapy such as anti-IgE, anti-TNF, anti-IL-5,anti-IL-6, anti-IL-12, anti-IL-1 and similar agents; receptor therapiese.g. entanercept and similar agents; antigen non-specificimmunotherapies (e.g. interferon or other cytokines/chemokines,cytokine/chemokine receptor modulators, cytokine agonists orantagonists, TLR agonists and similar agents).

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more other agents whichmay be useful in the prevention or treatment of cancer, for examplechemotherapeutics such as alkylating agents, topoisomerase inhibitors,antimetabolites, antimitotic agents, kinase inhibitors and similaragents; monoclonal antibody therapy such as trastuzumab, gemtuzumab andother similar agents; and hormone therapy such as tamoxifen, goserelinand similar agents.

The pharmaceutical compositions according to the invention may also beused alone or in combination with at least one other therapeutic agentin other therapeutic areas, for example gastrointestinal disease. Thecompositions according to the invention may also be used in combinationwith gene replacement therapy.

The invention includes in a further aspect a combination comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,together with at least one other therapeutically active agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with atleast one pharmaceutically acceptable diluent or carrier thereofrepresent a further aspect of the invention.

A therapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof will depend upon a number offactors. For example, the species, age, and weight of the recipient, theprecise condition requiring treatment and its severity, the nature ofthe composition, and the route of administration are all factors to beconsidered. The therapeutically effective amount ultimately should be atthe discretion of the attendant physician. Regardless, an effectiveamount of a compound of the present invention for the treatment ofhumans suffering from frailty, generally, should be in the range of0.0001 to 100 mg/kg body weight of recipient per day. More usually theeffective amount should be in the range of 0.001 to 10 mg/kg body weightper day. Thus, for a 70 kg adult one example of an actual amount per daywould usually be from 7 to 700 mg. For intranasal and inhaled routes ofadministration, typical doses for a 70 kg adult should be in the rangeof 1 microgramme to 1 mg per day. This amount may be given in a singledose per day or in a number (such as two, three, four, five, or more) ofsub-doses per day such that the total daily dose is the same. Aneffective amount of a pharmaceutically acceptable salt of a compound offormula (I) may be determined as a proportion of the effective amount ofthe compound of formula (I) or a pharmaceutically acceptable saltthereof per se. Similar dosages should be appropriate for treatment ofthe other conditions referred to herein.

Compounds of formula (I) and pharmaceutically acceptable salts thereofmay also be administered at any appropriate frequency e.g. 1-7 times perweek. The precise dosing regimen will of course depend on factors suchas the therapeutic indication, the age and condition of the patient, andthe particular route of administration chosen.

Pharmaceutical compositions may be presented in unit-dose formscontaining a predetermined amount of active ingredient per unit dose.Such a unit may contain, as a non-limiting example, 0.5 mg to 1 g of acompound of formula (I) or a pharmaceutically acceptable salt thereof,depending on the condition being treated, the route of administration,and the age, weight, and condition of the patient. Preferred unit-dosagecompositions are those containing a daily dose or sub-dose, as hereinabove recited, or an appropriate fraction thereof, of an activeingredient. Such pharmaceutical compositions may be prepared by any ofthe methods well-known in the pharmacy art.

There is thus further provided a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,and one or more pharmaceutically acceptable diluents or carriers.

There is also provided a process for preparing such a pharmaceuticalcomposition which comprises admixing a compound of formula (I), or apharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable diluents or carriers.

Throughout the description and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

The compounds of formula (I) and salts thereof may be prepared by themethodology described hereinafter, constituting further aspects of thisinvention.

Accordingly, there is provided a process for the preparation of acompound of formula (I), which process comprises the deprotection of acompound of formula (II):

wherein R¹, m, n, and p are as hereinbefore defined for a compound offormula (I) and R² is C₁₋₆alkyl, and thereafter, if required, carryingout one or more of the following optional steps:(i). removing any necessary protecting group;(ii). preparing a salt of the compound so-formed.

For example, A compound of formula (I) is dissolved in a suitablesolvent in the presence of a solution of a suitable acid, for example asolution of hydrogen chloride in 1,4-dioxane and stirred at a suitabletemperature, for example 35-40° C. for a suitable period of time, forexample 2-72 hours. The solvent is removed, for example under a streamof nitrogen, and the residue dissolved in a suitable solvent, forexample methanol, and loaded onto an ion-exchange cartridge, for examplean aminopropyl SPE cartridge. The cartridge is eluted with a suitablesolvent, for example methanol and the solvent removed to give a compoundof formula (I).

A compound of formula (II) may be prepared by reaction of a compound offormula (III), for example a salt of a compound of formula (III) such asthe trifluoroacetate salt:

wherein R¹ is as hereinbefore defined for a compound of formula (I) andR² is as hereinbefore defined for a compound of formula (II), with acompound of formula (IV):X—(CH₂)_(m)—X  (IV)wherein X is a leaving group, for example a halo group such as a bromogroup, followed by reaction with a compound of formula (V), or a salt ofa compound of formula (V) for example the hydrochloride salt in thepresence of a suitable base, for example aqueous sodium hydroxidesolution:

wherein n and p are as hereinbefore defined for a compound of formula(I).

For example, the trifluoroacetate salt of a compound of formula (III) isstirred with a suitable base, for example K₂CO₃, in a suitable solvent,for example DMF, for a suitable period of time, for example 1-2 hours,at a suitable temperature, for example at 45-55° C. The reaction mixtureis cooled and a compound of formula (IV) added. The reaction mixture isstirred at a suitable temperature, for example 18-25° C. for a suitableperiod of time, for example 35-45 minutes. The hydrochloride salt of acompound of formula (V) is shaken with a suitable base, for exampleaqueous NaOH solution, for a suitable period of time, for example 5-10minutes. A suitable organic solvent, for example DCM, is added and themixture passed through a phase separation cartridge. The solvent isremoved under mild condition, for example by using a rotary evaporator.The free base of the compound of formula (V) is added to the reactionmixture followed by a suitable base, for example triethylamine, and themixture stirred at a suitable temperature, for example 18-25° C. for asuitable period of time, for example 14-18 hours. Water is added and themixture extracted with a suitable solvent, for example DCM. The organicextract is separated using a hydrophobic frit and the solvent is thenremoved, for example under a stream of nitrogen. The crude product isthen purified by, for example, chromatography.

Alternatively, a compound of formula (II) may be prepared by reaction ofa compound of formula (VI):

wherein R¹ and m are as hereinbefore defined for a compound of formula(I), R² is as hereinbefore defined for a compound of formula (II), and Xis a leaving group, for example a halo group such as a bromo group, witha compound of formula (V), or a salt of a compound of formula (V) forexample the hydrochloride salt.

For example, a compound of formula (V) as its hydrochloride salt isshaken with a suitable base, for example aqueous NaOH, at a suitabletemperature, for example ambient temperature, for a suitable period oftime, for example 4-6 minutes. The free base of the compound of formula(V) is extracted with a suitable solvent, for example DCM, and thesolvent removed at low temperature, for example using a rotaryevaporator. The free base of the compound of formula (V) is then addedto a compound of formula (VI) in a suitable solvent, for example DMF,and a suitable base, for example triethylamine, is added. The mixture isstirred at a suitable temperature, for example 30-40° C. for a suitableperiod of time, for example 14-18 hours, then the hydrochloride salt ofa compound of formula (V) added and the reaction stirred at a suitabletemperature, for example 40-50° C. for a suitable period of time, forexample 14-18 hours, then an additional portion of the free base of acompound of formula (V) is added and the reaction stirred at a suitabletemperature, for example 30-40° C. for a suitable period of time, forexample 14-18 hours. Water is added and the mixture extracted with asuitable solvent, for example DCM through a hydrophobic frit. Thesolvent is then removed, for example under a stream of nitrogen. Thecrude product is then purified by, for example, chromatography.

Alternatively, a compound of formula (II), wherein n is 1 to 3, may beprepared by reaction of a compound of formula (VII):

wherein R¹ and m are as hereinbefore defined for a compound of formula(I) and R² is as hereinbefore defined for a compound of formula (II),with a compound of formula (VIII):

wherein n and p are as hereinbefore defined for a compound of formula(I), under conditions of reductive amination.

For example, to a compound of formula (VII) in a suitable solvent, forexample DCM, is added a compound of formula (VIII). A suitable reducingagent, for example NaBH(OAc)₃, and a suitable solvent, for example DCM,is added, followed by a suitable carboxylic acid, for example aceticacid. The mixture is stirred at a suitable temperature, for example28-25° C. under a suitable atmosphere, for example an atmosphere ofnitrogen, for a suitable period of time, for example 4-6 hours. Themixture is quenched with a suitable base, for example aqueous NaHCO₃,and diluted with a suitable solvent, for example DCM. The organic layerisolated, for example using a hydrophobic frit and the solvent removedunder reduced pressure. The crude product is then purified by, forexample, chromatography.

A compound of formula (VI) may be prepared by reaction of a compound offormula (III), for example a salt of a compound of formula (III) such asthe trifluoroacetate salt, with a compound of formula (IV).

For example, a mixture of the trifluoroacetate salt of a compound offormula (III) and a suitable base, for example potassium carbonate, in asuitable solvent, for example DMF, is heated at a suitable temperature,for example 45-55° C. under a suitable atmosphere, for example anatmosphere of nitrogen, for a suitable period of time, for example 1hour. The mixture is cooled to a suitable temperature, for exampleambient temperature, and a compound of formula (IV) added. The mixtureis heated at a suitable temperature, for example 45-55° C. for asuitable period of time, for example 14-18 hours. The mixture is cooledto a suitable temperature, for example ambient temperature, diluted withwater and extracted with a suitable solvent, for example DCM. Theorganic extracts are combined, isolated by, for example passing througha hydrophobic frit and evaporated to dryness to give a compound offormula (VI).

A compound of formula (VII) may be prepared by deprotection of acompound of formula (IX):

wherein R¹ and m are as hereinbefore defined for a compound of formula(I) and R² is as hereinbefore defined for a compound of formula (II).

For example, a compound of formula (IX) is dissolved in a suitablesolvent, for example ethanol, and hydrazine monohydrate is added and themixture stirred at a suitable temperature, for example ambienttemperature, for a suitable period of time, for example 8-12 hours. Thereaction mixture is then concentrated under reduced pressure and theresidue suspended in a suitable solvent, for example DCM, and stirredfor a suitable period of time, for example 1-2 hours. The suspension isfiltered, and the filtered solid washed with a suitable solvent, forexample DCM. The filtrate and washings are then washed with water anddried. The solution is concentrated under reduced pressure to give acompound of formula (VII).

A compound of formula (IX) may be prepared by reaction of a compound offormula (III) with a compound of formula (X):

wherein m is as hereinbefore defined for a compound of formula (I) and Xis a leaving group, for example a halo group such as a bromo group.

For example, a compound of formula (III) is dissolved in a suitablesolvent, for example DMF, and a suitable base, for example potassiumcarbonate, is added. The suspension is stirred at a suitabletemperature, for example room temperature, for a suitable period oftime, for example 15 minutes. A compound of formula (X) is added and thesuspension is vigorously stirred at a suitable temperature, for exampleroom temperature, for a suitable period of time, for example 18-12hours. The reaction mixture is extracted with a suitable solvent, forexample ethyl acetate, washed with water and brine, and dried. Thecompound of formula (IX) is obtained from the solvent, by, for example,crystallisation.

A salt of a compound of formula (III) may be prepared by deprotection ofa compound of formula (XI):

wherein R¹ is as hereinbefore defined for a compound of formula (I), R²is as hereinbefore defined for a compound of formula (II), and P is aprotecting group, for example a tetrahydro-2H-pyran-2-yl group, in thepresence of a suitable acid, for example trifluoroacetic acid.

For example, a suitable acid, for example trifluoroacetic acid, is addedto a solution of a compound of formula (XI) in a suitable solvent, forexample methanol. The mixture is stirred at a suitable temperature, forexample ambient temperature, for a suitable period of time, for example48-72 hours, to give a suspension. The reaction mixture is thenconcentrated under reduced pressure before being diluted with a suitablesolvent, for example ethyl acetate. The resultant mixture is filteredand washed with a small volume of a suitable solvent, for example ethylacetate until the filtrate is colourless. The residue is dried in airand then under reduced pressure to give the salt of a compound offormula (III). The filtrate may be concentrated and the concentratediluted with a small volume of a suitable solvent, for example ethylacetate, and then filtered and dried to yield a second crop of the saltof a compound of formula (III).

A compound of formula (III), for example a salt of a compound of formula(III) such as the trifluoroacetate salt, may also be prepared byreaction of a compound of formula (XII):

wherein R¹ is as hereinbefore defined for a compound of formula (I) andP is as hereinbefore defined for a compound of formula (XI), with asuitable halogenating agent, for example N-bromosuccinimide, followed byreaction with an alkoxide anion, for example a methoxide anion, and thenisolated in the presence of a suitable acid, for example trifluoroaceticacid.

For example, to a solution of crude compound of formula (XII) in asuitable dry solvent, for example dry chloroform, at a suitabletemperature, for example ambient temperature, is added a suitablehalogenating agent, for example N-bromosuccinimide, in portions over asuitable period of time, for example 5 minutes. The solution is stirredat a suitable temperature, for example ambient temperature, for asuitable period of time, for example 25-35 minutes. The reaction mixtureis then washed with water and the organic phase dried by, for example,passing through a hydrophobic frit and concentrated under reducedpressure. The resultant solid is dissolved in a suitable dry solvent,for example dry methanol, and a suitable alkoxide, for example asolution of sodium methoxide in methanol, is added at a suitabletemperature, for example ambient temperature, under an inert atmosphere,for example an atmosphere of nitrogen. The reaction mixture is heated ata suitable temperature, for example 60-70° C., with a condenserattached, for a suitable period of time, for example 12-18 hours. Thereaction mixture is then cooled and concentrated under reduced pressure.The residue is then taken up in a suitable solvent, for example ethylacetate, and poured into a suitable aqueous medium, for examplesaturated aqueous ammonium chloride solution. The organic layer isseparated and washed further with water, dried, for example overmagnesium sulphate, filtered and concentrated under reduced pressure. Toa solution of this material in a suitable dry solvent, such as drymethanol, at a suitable temperature, for example ambient temperature, isadded a suitable acid, for example trifluoroacetic acid. The reaction isstirred for a suitable period of time, for example 25-35 hours, andconcentrated under reduced pressure.

A compound of formula (XI) may be prepared by reaction of a compound offormula (XIII):

wherein R¹ is as hereinbefore defined for a compound of formula (I), Pis as hereinbefore defined for a compound of formula (XI), and Q is ahalogen atom, for example a bromine atom, with an alkoxide anion, forexample methoxide anion.

For example, a solution of a compound of formula (XIII) in a suitablesolvent, for example methanol, is heated to reflux with a solution of asuitable alkoxide, for example sodium methoxide, in a suitable solvent,for example methanol, for a suitable period of time, for example 4-5hours. The reaction mixture is concentrated under reduced pressure andpartitioned between a suitable organic solvent, for example ethylacetate, and a suitable aqueous medium, for example saturated aqueousammonium chloride solution. The organic phase is separated, washed, forexample with brine, and dried by, for example passing through ahydrophobic frit. The solvent is then removed under reduced pressure.

A compound of formula (XIII) may be prepared by reaction of a compoundof formula (XII) with a suitable halogenating agent, such asN-bromosuccinimide.

For example, a compound of formula (XII) is dissolved in a suitablesolvent, for example chloroform, and cooled to a suitable temperature,for example 0-0.5° C. To this solution is added a suitable halogenatingagent, such as N-bromosuccinimide, while maintaining the temperaturebelow about 3° C. The solution is stirred at a suitable temperature, forexample 2-3° C. for a suitable period of time, for example 30-45 minutesthen allowed to warm to a suitable temperature, for example ambienttemperature, and stirred for a suitable period of time, for example 5-7hours. The reaction mixture is then washed with water and the organicphase dried and separated from the aqueous phase using, for example, ahydrophobic frit. The organic solvent is then removed and the crudeproduct purified by, for example, chromatography.

A compound of formula (XII) wherein R¹ is C₁₋₆alkoxy may be prepared byreaction of a compound of formula (XIV):

wherein P is as hereinbefore defined for a compound of formula (XI), andT is a suitable leaving group, for example a halogen atom, for example afluorine atom or a chlorine atom, with a solution of a compound offormula (XV):R¹-M  (XV)wherein R¹ is C₁₋₆alkoxy and M is a suitable alkali metal ligand such assodium, prepared in a solvent of formula (XVS):R¹—H  (XVS)wherein the R¹ group in the compound of formula (XV) is the same as theR¹ group in the solvent of formula (XVS).

For example, a compound of formula (XV) such as sodium t-butoxide, isadded to a solvent of formula (XVS). The mixture is stirred untilhomogeneous, then a compound of formula (XIV) is added. The reactionmixture is heated to a suitable temperature, for example 50-100° C., fora suitable period of time, for example 12-18 hours. The solvent issubstantially removed under reduced pressure and partitioned between asuitable solvent, for example diethyl ether, and water. The organicphase is separated and the aqueous phase re-extracted with furthersolvent. The organic layers are then isolated, combined, dried using asuitable drying agent, for example anhydrous magnesium sulphate. Thedrying agent is removed by filtration and the solvent removed from theproduct under reduced pressure.

A compound of formula (XII) wherein R¹ is C₁₋₆alkylamino may be preparedby reaction of a compound of formula (XIV) with a compound of formula(XVI):R¹—H  (XVI)wherein R¹ is C₁₋₆alkylamino.

For example, a compound of formula (XIV) is added to a solution of acompound of formula (XVI) in a suitable dry solvent, for example dryethylene glycol, at a suitable temperature, for example ambienttemperature, under a suitable inert atmosphere, for example anatmosphere of nitrogen. The reaction mixture is heated at a suitabletemperature, for example 110-130° C., for a suitable period of time, forexample 12-18 hours. The reaction is then cooled to a suitabletemperature, for example ambient temperature, diluted with a suitablesolvent, for example ethyl acetate, and washed with water. The organiclayer is dried with a suitable drying agent, for example anhydrousmagnesium sulphate, filtered and concentrated under reduced pressure toyield a compound of formula (XII) wherein R¹ is C₁₋₆alkylamino.

A compound of formula (XIV) may be prepared by reaction of a compound offormula (XVII):

wherein P is as hereinbefore defined for a compound of formula (XI), andT is as hereinbefore defined for a compound of formula (XIV), and V is asuitable leaving group, for example a halogen atom, for example achlorine atom, with an alcoholic solution of ammonia, for example asolution of ammonia in iso-propyl alcohol.

For example, a compound of formula (XVII) is heated with an alcoholicsolution of ammonia, for example a 2M solution of ammonia in iso-propylalcohol, at a suitable temperature, for example 50-60° C., for asuitable period of time, for example 5-6 hours. The reaction mixture isthen left to stand at a suitable temperature, for example ambienttemperature, for a suitable period of time, for example 12-18 hours. Afurther quantity of the alcoholic solution of ammonia, for example a 2Msolution of ammonia in iso-propyl alcohol, is added to break up theresultant cake and the reaction mixture heated for a further period oftime, for example 8-10 hours, until the reaction is complete. Water isadded to the reaction mixture and the solid removed by filtration,washed with a suitable washing medium, for example a mixture ofiso-propyl alcohol and water, and then dried, for example by air-dryingunder suction to give a first crop of a compound of formula (XIV). Thefiltrate is allowed to stand for a further period of time, for example12-18 hours and the resultant second crop of a compound of formula (XIV)isolated by filtration and dried.

A compound of formula (XIV) may also be prepared by reaction of acompound of formula (XVIII):

wherein T is as hereinbefore defined for a compound of formula (XIV),and V is as hereinbefore defined for a compound of formula (XVII), witha compound of formula (XIX):P^(U)—H  (XIX)wherein P^(U) is a suitable precursor to the protecting group P, forexample a 3,4-dihydro-2H-pyranyl group, followed by reaction with analcoholic solution of ammonia, for example a solution of ammonia iniso-propyl alcohol.

For example, p-toluenesulfonic acid monohydrate is added to a solutionof a compound of formula (XVIII) in a suitable dry solvent, for exampledry ethyl acetate. The reaction mixture is heated to a suitabletemperature, for example 50-60° C., and a compound of formula (XIX)added. The reaction is stirred at a suitable temperature, for example50-60° C., for a suitable period of time, for example 1-2 hours, and thesolvent removed under reduced pressure. A suspension of the resultantsolid in an alcoholic solution of ammonia, for example a 2M solution ofammonia in iso-propyl alcohol is heated under a suitable inertatmosphere, for example an atmosphere of nitrogen, at a suitabletemperature, for example 60-70° C., for a suitable period of time, forexample 4-5 hours with an attached condenser. The reaction mixture ispoured into water and allowed to cool for a suitable period of time, forexample 12-18 hours. The resultant precipitate is isolated by filtrationand dried.

A compound of formula (XIV) may also be prepared by reaction of acompound of formula (XVIIIA):

wherein T is a fluorine atom, with a suitable protecting agent, forexample a silylating agent such as N,O-bis(trimethylsilyl)acetamide,followed by reaction of the protected compound of formula (XVIII) with acompound of formula (XIXE):P^(U)-E  (XIXE)wherein P^(U) is a suitable precursor to the protecting group P, forexample a 3,4-dihydro-2H-pyranyl group and E is an acetyloxy group, forexample an acetate group.

For example, N,O-bis(trimethylsilyl)acetamide is added to a stirredsuspension of a compound of formula (XVIII) in a suitable anhydroussolvent, for example anhydrous acetonitrile and the resulting mixtureheated to reflux and maintained at that temperature for a suitableperiod of time, for example 1-3 hours. The reaction mixture is thencooled to a suitable temperature, for example 0-5° C. A solution of acompound of formula (XIXE) in a suitable anhydrous solvent, for exampleanhydrous acetonitrile is then added slowly, followed by dropwiseaddition of a Lewis acid, for example trimethylsilyltrifluoromethanesulfonate. The reaction mixture is warmed to a suitabletemperature, for example 8 to 15° C. and stirring for a further periodof time, for example 1-2 hours. The mixture is then quenched by additionof a suitable base, for example 1M sodium carbonate. Additional water isthen added to the suspension. The majority of the aqueous layer andinorganic solid was separated. The organic layer is cooled to 0-5° C.with stirring to encourage further precipitation. The solid was thecollected by filtration and dried.

Abbreviations

The following list provides definitions of certain abbreviations as usedherein. It will be appreciated that the list is not exhaustive, but themeaning of those abbreviations not herein below defined will be readilyapparent to those skilled in the art.

-   DCM Dichloromethane-   DME 1,2-Dimethoxyethane-   DMF N,N-Dimethylformamide-   EtOAc Ethyl acetate-   Et₂O Diethyl ether-   h hours-   HCl Hydrochloric acid-   HPLC High performance liquid chromatography-   ISCO Companion Automated flash chromatography equipment with    fraction analysis by UV absorption available from Presearch Limited,    Basingstoke, Hants., RG24 8PZ, UK-   MDAP HPLC Reverse phase HPLC on a C₁₈ column using a two-solvent    gradient and analysis of the fractions by electrospray mass    spectroscopy.-   MeOH Methanol-   mins minutes-   NBS N-Bromosuccinimide-   Stripped Removal of solvent under reduced pressure-   TBME Tertiary butyl methyl ether-   TFA Trifluoroacetic acid-   iPr iso-Propyl-   t-Bu tert-Butyl-   Ms Mesyl-   Ac Acetyl-   n-Bu n-Butyl-   Ph Phenyl

The synthetic processes hereinbefore described are summarised in Scheme1.

Typical reaction conditions for each of the synthetic steps of Scheme 1are provided below:

-   A Dihydropyran/paratoluene sulphonic acid, e.g. 50° C. for 3-6    hours.-   A1 Dihydropyran/paratoluene sulphonic acid, e.g. 50° C. for 1 hour,    then ammonia/iPrOH, e.g. 60° C. for 4 hours, then add water and cool    to ambient temperature over 12-18 hours.-   A2 BSA in MeCN, reflux, cool to 0° C., then THP acetate in MeCN,    warm to 10° C., then NaHCO₃ (aq.)-   B Ammonia/iPrOH, e.g. 50° C. for 5 hours, then ambient temperature    for 12-18 hours, then 50° C. for 9 hours.-   C For X═NH(R^(A)═C₁₋₆alkyl). R^(A)NH₂/ethylene glycol e.g. 120° C.    for 12-18 hours. For X═O. R^(A)OH(R^(A)═C₁₋₆alkyl).    NaOtBu/dimethoxyethane e.g. 93-110° C. for 12-18 hours.-   C1 NBS in CHCl₃ e.g. 0-5° C. for 30 minutes then ambient temperature    for 0.5-1 hour, NaOMe/MeOH, e.g. 65° C., 12-18 hours, then TFA/MeOH    e.g. ambient temperature for 18-65 hours.-   D NBS in CHCl₃ e.g. 0-5° C. for 30 minutes then ambient temperature    for 36-48 hours.-   E NaOMe/MeOH e.g. reflux 4-6 hours.-   F TFA/MeOH e.g. ambient temperature for 18-65 hours.-   G K₂CO₃, DMF, 50° C., 1 hour then e.g. 1,3-dibromopropane, 20° C. 40    mins., then amine of formula (V) and Et₃N at r.t., or;    -   K₂CO₃, DMF, 50° C., 1 hour then e.g. 1,3-dibromopropane, 20° C.        40 mins., then amine of formula (V) and Barton's base at r.t.-   G1 K₂CO₃/DMF, 1,3-dibromopropane, DMF, 50° C. overnight.-   G2 Amine of formula (V), Et₃N, DMF, 35-45° C., overnight.-   G3 K₂CO₃/DMF, then compound of formula (X), r.t. for 10 hr.-   G4 NH₂.NH₂, H₂O, EtOH, r.t. 10 hr.-   G5 Compound of formula (VIII), NaBH(OAc)₃, DCM, r.t. 5 hr.-   H HCl/dioxane, then ambient temperature for 18 hours.

Compounds of formulae (IV), (V), (VIII), (X), (XII), (XIV), (XV), (XVI),(XVII), (XVIII), and (XVIIIA) are commercially available, for examplefrom Sigma-Aldrich, UK, or may be prepared by analogy with knownprocedures, for example those disclosed in standard reference texts ofsynthetic methodology such as J. March, Advanced Organic Chemistry, 6thEdition (2007), WileyBlackwell, or Comprehensive Organic Synthesis(Trost B. M. and Fleming I., (Eds.), Pergamon Press, 1991), eachincorporated herein by reference as it relates to such procedures.

Examples of other protecting groups that may be employed in thesynthetic routes described herein and the means for their removal can befound in T. W. Greene ‘Protective Groups in Organic Synthesis’, 4thEdition, J. Wiley and Sons, 2006, incorporated herein by reference as itrelates to such procedures.

For any of the hereinbefore described reactions or processes,conventional methods of heating and cooling may be employed, for exampletemperature-regulated oil-baths or temperature-regulated hot-blocks, andice/salt baths or dry ice/acetone baths respectively. Conventionalmethods of isolation, for example extraction from or into aqueous ornon-aqueous solvents may be used. Conventional methods of drying organicsolvents, solutions, or extracts, such as shaking with anhydrousmagnesium sulphate, or anhydrous sodium sulphate, or passing through ahydrophobic frit, may be employed. Conventional methods of purification,for example crystallisation and chromatography, for example silicachromatography or reverse-phase chromatography, may be used as required.Crystallisation may be performed using conventional solvents such asethyl acetate, methanol, ethanol, or butanol, or aqueous mixturesthereof. It will be appreciated that specific reaction timestemperatures may typically be determined by reaction-monitoringtechniques, for example thin-layer chromatography and LC-MS.

Where appropriate individual isomeric forms of the compounds of theinvention may be prepared as individual isomers using conventionalprocedures such as the fractional crystallisation of diastereoisomericderivatives or chiral high performance liquid chromatography (chiralHPLC).

The absolute stereochemistry of compounds may be determined usingconventional methods, such as X-ray crystallography.

Aspects of the invention are illustrated by reference to, but are in noway limited by, the following Examples.

General Experimental Details

Compounds were named using ACD/Name PRO 6.02 chemical naming softwarefrom Advanced Chemistry Developments Inc., Toronto, Ontario, M5H2L3,Canada.

Experimental details of LCMS systems A-H as referred to herein are asfollows:

System A

Column: 50 mm × 2.1 mm ID, 1.7 μm Acquity UPLC BEH C₁₈ Flow Rate: 1mL/min. Temp: 40° C. UV detection range: 210 to 350 nm Mass spectrum:Recorded on a mass spectrometer using alternative-scan positive andnegative mode electrospray ionisation Solvents: A: 0.1% v/v formic acidin water B: 0.1% v/v formic acid acetonitrile Gradient: Time (min.) A %B % 0 97  3 1.5  0 100 1.9  0 100 2.0 97  3System B

Column: 30 mm × 4.6 mm ID, 3.5 μm Sunfire C₁₈ column Flow Rate: 3mL/min. Temp: 30° C. UV detection range: 210 to 350 nm Mass spectrum:Recorded on a mass spectrometer using alternative-scan positive andnegative mode electrospray ionisation Solvents: A: 0.1% v/v solution offormic acid in water B: 0.1% v/v solution of formic acid acetonitrileGradient: Time (min.) A % B % 0 97  3 0.1 97  3 4.2  0 100 4.8  0 1004.9 97  3 5.0 97  3System C

Column: 50 mm × 2.1 mm ID, 1.7 μm Acquity UPLC BEH C₁₈ Flow Rate: 1mL/min. Temp: 40° C. UV Detection Range: 210 to 350 nm Mass spectrum:Recorded on a mass spectrometer using alternative-scan positive andnegative mode electrospray ionisation Solvents: A: 10 mM ammoniumbicarbonate in water adjusted to pH 10 with ammonia solution B:acetonitrile Gradient: Time (min.) A % B % 0 99  1 1.5  3  97 1.9  3  972.0  0 100System D

Column: 50 mm × 4.6 mm ID, 3.5 μm XBridge C₁₈ column Flow Rate: 3mL/min. Temp: 30° C. UV Detection Range: 210 to 350 nm Mass spectrum:Recorded on a mass spectrometer using alternative-scan positive andnegative mode electrospray ionisation Solvents: A: 10 mM ammoniumbicarbonate in water adjusted to pH 10 with ammonia solution B:acetonitrile Gradient: Time (min.) A % B % 0 99  1 0.1 99  1 4.0  3 975.0  3 97

Chromatographic purification was typically performed using pre-packedsilica gel cartridges. The Flashmaster II is an automated multi-userflash chromatography system, available from Argonaut Technologies Ltd,which utilises disposable, normal phase, Solid Phase Extraction (SPE)cartridges (2 g to 100 g). It provides quaternary on-line solvent mixingto enable gradient methods to be run. Samples are queued using themulti-functional open access software, which manages solvents,flow-rates, gradient profile and collection conditions. The system isequipped with a Knauer variable wavelength UV-detector and two GilsonFC204 fraction-collectors enabling automated peak cutting, collectionand tracking.

Solvent removal using a stream of nitrogen was performed at 30-40° C. ona GreenHouse Blowdown system available from Radleys DiscoveryTechnologies Saffron Walden, Essex, CB11 3AZ. UK

¹H NMR spectra were recorded in either CDCl₃ or DMSO-d₆ on either aBruker DPX 400 or Bruker Avance DRX or Varian Unity 400 spectrometer allworking at 400 MHz. The internal standard used was eithertetramethylsilane or the residual protonated solvent at 7.25 ppm forCDCl₃ or 2.50 ppm for DMSO-d₆.

Mass directed autopreparative HPLC was undertaken under the conditionsgiven below. The UV detection was an averaged signal from wavelength of210 nm to 350 nm and mass spectra were recorded on a mass spectrometerusing alternate-scan positive and negative mode electrospray ionization.

Method A:

Method A was conducted on an XBridge C₁₈ column (typically 150 mm×19 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=10 mM aqueous ammonium bicarbonate adjusted to pH 10 with ammoniasolution.

B=acetonitrile.

Method B:

Method B was conducted on a Sunfire C₁₈ column (typically 150 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=0.1% v/v solution of formic acid in water

B=0.1% v/v solution of formic acid in acetonitrile.

Method C:

Method C was conducted on a Sunfire C₁₈ column (typically 150 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=0.1% v/v solution of trifluoroacetic acid in water

B=0.1% v/v solution of trifluoroacetic acid in acetonitrile.

Method D:

Method D was conducted on an Atlantis C₁₈ column (typically 100 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=0.1% v/v solution of formic acid in water

B=0.1% v/v solution of formic acid in acetonitrile.

EXAMPLES Intermediate 1:2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine

To 2,6-dichloropurine (25.0 g) (available from, for example, Aldrich,UK) was added ethyl acetate (260 mL), followed by p-toluenesulfonic acid(0.253 g). The mixture was heated to 50° C. and then3,4-dihydro-2H-pyran (16.8 g) was added. The reaction mixture was thenheated at 50° C. for 4 hours. The reaction mixture was evaporated invacuo to give the title compound as a yellow solid (36.9 g).

1H NMR (CDCl₃): 8.35 (1H, s), 5.77 (1H, dd), 4.20 (1H, m), 3.79 (1H, m),2.20-1.65 (6H, m).

Intermediate 2: 2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (36.9 g) was heatedwith 2M ammonia in isopropanol (250 mL) at 50° C. for 5 hours. Afterstanding at ambient temperature overnight, a further quantity of 2Mammonia in isopropanol (100 mL) was added to break up the resultant cakeand the reaction mixture was heated for a further 9 hours until thereaction was complete. To the reaction mixture was added water (70 mL)and the yellow solid filtered off. The solid was washed with isopropylalcohol:water (5:1 (v/v), 60 mL) and then air-dried under suction togive a first crop. The filtrate was re-filtered after standing overnightto isolate precipitate and both solids were dried in vacuo. The firstcrop was pure with the second crop material showing a very minorimpurity (isolated broad signal 3.5 ppm not seen in first crop) but wasotherwise identical. Solid first crop (28.4 g), solid second crop (3.42g).

1H NMR (CDCl₃): 8.01 (1H, s), 5.98 (2H, broad s), 5.70 (1H, dd), 4.16(1H, m), 3.78 (1H, m), 2.15-1.60 (6H, overlapping m).

Intermediate 2 (alternative method):2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

To a solution of 2,6-dichloropurine (25 g) (available from, for example,Aldrich, UK) in dry ethyl acetate (200 ml) was added p-toluenesulfonicacid monohydrate (235 mg). The reaction was heated to 50° C. and3,4-dihydro-2H-pyran (18.1 ml) was added in one go. The reaction wasallowed to stir at 50° C. for 1 hour and the solvent was removed underreduced pressure. This afforded a yellow solid. A suspension of thissolid (˜36 g) in 2.0M ammonia in isopropanol (460 ml) was heated undernitrogen at 60° C. for 4 hours with an attached condenser. The reactionwas poured into water (50 ml) and left to cool overnight. Theprecipitate was filtered and dried on a rotary evaporator (60° C.) for30 minutes to afford the title compound as an off-white solid, 31 g(93%, 2 steps).

MS calcd for (C₁₀H₁₂ClN₅O)⁺=254, 256

MS found (electrospray): (M)⁺=254, 256 (3:1)

¹H NMR ((CD₃)₂SO): δ 8.43 (1H, s), 7.82 (2H, s), 5.55 (1H, dd), 4.00(1H, m), 3.69 (1H, m), 2.21 (1H, m), 1.95 (2H, m), 1.74 (1H, m), 1.56(2H, m).

Intermediate 3: 2-Butoxy-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

To butan-1-ol (76 mL) was added portion-wise sodium tert-butoxide (15.2g) (Note: reaction mixture gets warm). The above was stirred untilhomogeneous (˜15 min) before2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (10.0 g) was thenadded to the resultant pale yellow solution. The reaction mixture wasthen heated to 100° C., overnight. The reaction mixture was stripped toremove as much butan-1-ol as possible before being partitioned betweendiethyl ether and water. The diethyl ether phase was separated and theaqueous re-extracted further with diethyl ether. Combined organic layersdried over magnesium sulphate (anhydrous). Magnesium sulphate wasfiltered off and filtrate stripped to give brown viscous oil which wasazeotroped with toluene (3 times) and placed under high vacuumovernight, transferred to new flask with dichloromethane and stripped,placed under high vacuum to give the title compound as a brown glass(9.45 g).

1H NMR (CDCl₃): 7.85 (1H, s), 5.92 (2H, broad s), 5.64 (1H, d), 4.32(2H, t), 4.14 (1H, m), 3.75 (1H, m), 2.10-1.95 (3H, overlapping m),1.81-1.58 (5H, overlapping m), 1.50 (2H, m), 0.97 (3H, t).

Intermediate 4:8-Bromo-2-butoxy-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

2-(Butoxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (9.45 g) wasdissolved in chloroform (50 mL) and cooled to 0° C. (ice-bath). To thissolution was added portion wise N-bromosuccinimide (6.07 g) keeping thetemperature below 3° C. This gave a dark green solution, stirred at 2.5°C. for 30 minutes before allowing to warm to room temperature and thenstirring for 6 hours. The reaction mixture was then washed with water(100 mL, twice). Organic phase was dried/separated using a hydrophobicfrit and evaporated to give a dark brown gum which was purified bysilica chromatography (120 g) (ISCO) using a gradient elution of 0-50%ethyl acetate:cyclohexane to afford the title compound as a pale yellowsolid (8.37 g).

1H NMR (CDCl₃): 5.61 (1H, dd), 5.49 (2H, broad s), 4.32 (2H, m), 4.17(1H, m), 3.71 (1H, m), 3.04 (1H, m), 2.11 (1H, broad d), 1.89-1.45 (6H,overlapping m), 1.50 (2H, m), 0.97 (3H, t).

Intermediate 5:2-Butoxy-8-methoxy-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

8-Bromo-2-(butoxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (8.37g) was heated to reflux with 25% sodium methoxide in methanol (14.4 mL)and methanol (65 mL) for 4.5 hours. The reaction mixture wasconcentrated under reduced pressure and partitioned between ethylacetate and saturated ammonium chloride solution. Separated organicphase and repeated extraction into ethyl acetate. Combined organicphases and washed with brine (twice). The organic phase was passedthrough a hydrophobic frit after separating aqueous and was evaporatedto give a light brown gum which was placed under high vacuum to give afoam (7.52 g) which collapsed to a gum (7.34 g) at ambient pressure andsolidified overnight to give the title compound as a yellow amorphoussolid.

MS calcd for (C₁₅H₂₃N₅O₃)⁺=321

MS found (electrospray): (M+H)⁺=322

1H NMR (CDCl₃): 5.50 (1H, dd), 5.17 (2H, broad s), 4.29 (2H, t), 4.12(3H, s and 1H, m), 3.70 (1H, m), 2.77 (1H, m), 2.05 (1H, m), 1.82-1.63(6H, overlapping m), 1.50 (2H, m), 0.97 (3H, t).

Intermediate 6: 2-Butoxy-8-methoxy-9H-purin-6-amine trifluoroacetatesalt

To a solution of2-(butoxy)-8-(methoxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(7.34 g) in methanol (100 mL) was added trifluoroacetic acid (10 mL).The mixture was stirred at ambient temperature over the weekend to givea suspension. The reaction mixture was concentrated to a small volume(thick slurry) before being diluted with ethyl acetate (50 mL). Theresultant slurry was filtered and washed with a small volume of ethylacetate until the filtrate was colourless. The solid remaining was driedby air and then in vacuo to give the title compound as a white solid(6.20 g). The filtrate obtained previously was concentrated to give aslurry which was diluted with a small volume of ethyl acetate (10 mL)and then filtered and dried as above. This second crop was isolated as awhite solid (0.276 g). Both crops were identical by NMR.

MS calcd for (C₁₀H₁₅N₅O₂)⁺=237

MS found (electrospray): (M+H)⁺=238

1H NMR (CD₃OD): 4.47 (2H, t), 4.15 (3H, s), 1.80 (2H, m), 1.50 (2H, m),0.99 (3H, t) (exchangeable NH₂, NH and COOH protons not observed).

Intermediate 7:N²-Butyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

To a solution of 2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(10 g) in dry ethylene glycol (50 ml) at room temperature and undernitrogen was added n-butylamine (16 ml) in one go. The reaction washeated at 120° C. overnight. The reaction was cooled to roomtemperature, diluted with ethyl acetate (150 ml) and washed with water(2×50 ml). The organic layer was dried over MgSO₄, filtered andconcentrated in vacuo. This afforded the title compound as a viscousgreen oil (10.2 g) that was used in the next step without furtherpurification.

MS calcd for (C₁₄H₂₂N₆O)⁺=290

MS found (electrospray): (M+H)⁺=291

¹H NMR ((CD₃)₂SO): δ 7.8 (1H, s), 6.6 (2H, s), 6.2 (1H, t), 5.4 (1H,dd), 4.0 (1H, m), 3.6 (1H, m), 3.2 (2H, m), 2.2 (1H, m), 1.9 (1H, m),1.8 (1H, m), 1.7 (1H, m), 1.5 (2H, m), 1.4 (2H, m), 1.3 (2H, m), 0.9(3H, t).

Intermediate 8: N²-Butyl-8-methoxy-9H-purine-2,6-diamine trifluoroaceticacid salt

To a solution of crudeN²-butyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine (˜10.2 g) indry chloroform (100 ml) at room temperature was added N-bromosuccinimide(6.3 g) in portions over 5 minutes. The dark solution was allowed tostir at room temperature for 30 minutes. The reaction mixture was washedwith water (20 ml). The organic phase was passed through a hydrophobicfrit and concentrated in vacuo. This afforded a beige solid which wasdissolved in dry methanol (100 ml) and at room temperature undernitrogen was added sodium methoxide solution (25 wt. % in methanol, 24ml) in one go. The reaction was heated at 65° C., with a condenserattached, overnight. The reaction was cooled and concentrated in vacuo.The resultant orange residue was taken up in ethyl acetate (150 ml) andpoured into saturated aqueous ammonium chloride (50 ml). The organiclayer was separated and washed further with water (50 ml). The organiclayer was dried over MgSO₄, filtered and concentrated in vacuo. To thismaterial in dry methanol (70 ml) at room temperature was addedtrifluoroacetic acid (7 ml) in one go. The reaction was stirred for 30hours and concentrated in vacuo to yield a dark brown solid. This wastaken up in diethyl ether (20 ml) and triturated. The solid was filteredto afford the title compound as a beige solid (3.3 g, 35%, 4 steps).

MS calcd for (C₁₀H₁₆N₆O)⁺=236

MS found (electrospray): (M+H)⁺=237

¹H NMR ((CD₃)₂SO): δ 13.3-12.3 (1H, br.m), 8.6-7.3 (2H, m), 4.05 (3H,s), 3.28 (2H, m), 1.52 (2H, m), 1.33 (2H, m), 0.89 (3H, t) (remainingexchangeable protons not clear).

Intermediate 9:2-{[(1S)-1-Methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Method A

Sodium t-butoxide (48.5 g, 505 mmol) was added portionwise to(S)-2-pentanol (available from, for example, Julich Chiral Solutions,Germany) (185 ml) at room temperature and stirred until homogeneous(Note reaction is exothermic).2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (32 g, 126 mmol)was added and the reaction mixture heated at 70° C. for 72 hours. Thereaction was cooled to room temperature and partitioned between ethylacetate (500 ml) and water (500 ml). The organic phase was washed withsaturated sodium chloride solution (100 ml), dried (MgSO₄), filtered andevaporated. The residue was triturated with ether and the solid materialfiltered. The precipitate was re-washed with ether and the filtratescombined and evaporated. The crude material (ca. 30 g) was dissolved inDMSO:methanol (1:1) and purified by chromatography on a reverse phase(C₁₈) column (330 g) using a gradient of 25-65% acetonitrile (+0.1%TFA)-water (+0.1% TFA) over 8 column volumes, the fractions wereimmediately neutralised with saturated aqueous sodium carbonatesolution. Appropriate fractions were combined and partitioned betweendichloromethane and saturated aqueous sodium hydrogen carbonate. Theorganic phase was dried by passage through a hydrophobic frit, filteredand evaporated to give the title compound as a pale cream foam (14.97g).

LCMS (System B): t_(RET)=2.21 min; MH⁺ 306

Method B

Sodium t-butoxide (206 g, 2.144 mol) was added to (S)-2-pentanol(available from, for example, Julich Chiral Solutions, Germany) (720 ml,6.58 mol) in a 2 L round bottomed flask. The mixture was stirred and 50°C. until all the sodium t-butoxide had dissolved.2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (130 g, 548 mmol)was then added in portions over 5 minutes. After 3 hours LCMS analysisindicated complete consumption of the starting material and the mixturewas poured into ice/water (3 L) and then extracted with methyl t-butylether. This resulted in emulsion formation and the mixture was filteredthrough Celite and the organic phase was separated. The aqueous layerwas then treated with solid NaCl and then re-extracted with methylt-butyl ether. The organic extracts were combined and washed with brine,dried over magnesium sulfate, filtered and then evaporated to yield thetitle compound as a pale brown gum (158.59 g).

LCMS (System D): t_(RET)=2.65 min; MH⁺ 306

Intermediate 10:8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-bromosuccinimide (12.16 g, 68.3 mmol) was added portionwise over 5min. to a stirred solution of2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(14.9 g, 48.8 mmol) in chloroform (80 ml) at <5° C. under an atmosphereof nitrogen. The reaction mixture was stirred at <5° C. for 5 hours thenwashed with saturated sodium hydrogen carbonate solution (80 ml) thenwater (80 ml) and the solvent removed. The residual foam was dissolvedin DCM (50 ml) and washed with water (50 ml) then brine (50 ml). Thecombined aqueous phases were washed with DCM (50 ml). The combinedorganic layers were dried through a hydrophobic frit, and the solventremoved in vacuo to yield the title compound as an orange foam (18.5 g).

LCMS (System D): t_(RET)=3.06 min; MH⁺ 384/386

Intermediate 11:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(7.1 g, 18.48 mmol) was dissolved in anhydrous methanol (70 mL) and asolution of sodium methoxide (25%) in methanol (8 ml) was added dropwiseunder an atmosphere of nitrogen. The solution was heated to reflux at90° C. for 4 hours under an atmosphere of nitrogen. Additional sodiummethoxide in methanol (25% solution, 3 ml) was added and the reactionwas stirred at 60° C. for a further 16 hours. An additional portion ofsodium methoxide in methanol (25% solution, 5 ml) was added and thereaction was stirred at 90° C. for a further 7 hours. The solvent wasremoved on the rotary evaporator and the crude product was partitionedbetween EtOAc (75 ml) and saturated ammonium chloride solution (75 ml).The organic layer was washed with brine (75 ml). The solvent was removedon the rotary evaporator to yield the title compound as a pale orangefoam (6 g).

LCMS (System D): t_(RET)=3.08 min; MH⁺ 336

Intermediate 12:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate

2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(6 g, 17.89 mmol) was dissolved in methanol (50 ml). Trifluoroaceticacid (20.67 ml, 268 mmol) was added dropwise, and the mixture stirred at20° C. for 72 hours under an atmosphere of nitrogen. The solvent wasremoved in vacuo, and the resulting solid was washed with ethyl acetateand filtered. The filtrate was stripped and the residue washed withethyl acetate. The combined solid residues were dried in the vacuum ovenfor 2 hours to give the title compound as an off-white solid (5.3 g).

LCMS (System C): t_(RET)=0.76 min; MH⁺ 252

Intermediate 13:2-(Butyloxy)-8-(methyloxy)-9-{3-[(2R)-tetrahydro-2-furanylamino]propyl}-8,9-dihydro-7H-purin-6-amine

2-(Butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate (70 mg,0.199 mmol) was stirred with K₂CO₃ (68.9 mg, 0.498 mmol) in DMF (1 ml)for 1 hour at 50° C. The reaction mixture was cooled and1,3-dibromopropane (0.020 ml, 0.199 mmol) was added. The reactionmixture was stirred at 20° C. for 40 mins. (3S)-Tetrahydro-3-furanaminehydrochloride (49.3 mg, 0.399 mmol) was shaken with aqueous NaOHsolution (0.996 ml, 1.993 mmol) for 5 mins., DCM, (2×3 ml) was added andthe mixture passed through a phase separation cartridge. The solvent wasremoved under mild conditions by rotary evaporation. The free base wasadded to the reaction mixture followed by triethylamine (0.056 ml, 0.399mmol) and the mixture was stirred at 20° C. for 16 hours.(3S)-Tetrahydro-3-furanamine hydrochloride (49.3 mg, 0.399 mmol) wasshaken with NaOH (0.996 ml, 1.993 mmol) for 5 mins., DCM (2×3 ml) wasadded, and the mixture passed through a phase separation cartridge andadded directly to the reaction mixture. Triethylamine (0.056 ml, 0.399mmol) was added and the mixture was stirred at 20° C. for 16 hours.Water was added and the mixture extracted with DCM (2×5 ml) through ahydrophobic frit. The solvent was removed by nitrogen blowdown. Thesamples were dissolved in 1:1 MeOH:DMSO (1 ml) and purified by MassDirected AutoPrep (Method A). The solvent was dried under a stream ofnitrogen to give the title compound as a white solid (3 mg).

LCMS (System D): t_(RET)=2.14 min; MH⁺ 365

Intermediate 14:2-(Butyloxy)-8-(methyloxy)-9-{3-[(2S)-tetrahydro-2-furanylamino]propyl}-9H-purin-6-amine

2-(Butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate (100 mg,0.285 mmol) and K₂CO₃ (98 mg, 0.712 mmol) was stirred in DMF (2 ml) at50° C. for 1 hour. The reaction was cooled and 1,3-dibromopropane (0.029ml, 0.285 mmol) was added. The mixture was stirred at 20° C. for 40mins. (3R)-Tetrahydro-3-furanamine hydrochloride (35.2 mg, 0.285 mmol)and N″-(1,1-dimethylethyl)-N,N,N′,N′-tetramethylguanidine (146 mg, 0.854mmol) were stirred in DMF 1 ml at 20° C. for 40 mins. The resultantmixture of assumed free base was added to the first reaction vessel andthe mixture stirred for 16 hours at 20° C. Water (2 ml) and brine (2 ml)were added to the mixture. The product was extracted with DCM (2×5 ml)using a hydrophobic separation cartridge and the solvent removed on theN₂ blowdown unit. The sample was dissolved in 1:1 MeOH:DMSO (1 ml) andpurified by Mass Directed AutoPrep (Method A). The solvent was driedunder a stream of nitrogen to give the title compound as a white solid(7 mg).

LCMS (System D): t_(RET)=2.15 min; MH⁺ 365

Intermediate 15:2-(Butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-9H-purin-6-amine

2-(Butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate (70 mg, 0.2mmol) and K₂CO₃ (68.8 mg, 0.050 mmol) was stirred in DMF (2 ml) at 50°C. for 1 hour. The reaction was cooled and 1,3-dibromopropane (0.020 ml,0.2 mmol) was added. The mixture was stirred at 20° C. for 40 mins.(Tetrahydro-2-furanylmethyl)amine (0.41 ml, 0.2 mmol) and triethylamine(0.056 ml, 0.4 mmol) in DMF (1 ml) was added and the reaction mixturestirred at rt for 16 hours. Water (2 ml) and brine (2 ml) were added tothe mixture. The product was extracted with DCM (2×5 ml) using ahydrophobic separation cartridge and the solvent removed on the N₂blowdown unit. The sample was dissolved in 1:1 MeOH:DMSO (1 ml) andpurified by Mass Directed AutoPrep (Method A). The solvent was driedunder a stream of nitrogen to give the title compound as a white solid(20 mg).

LCMS (System D): t_(RET)=2.35 min; MH⁺ 379

Intermediate 16:2-(Butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-3-furanylmethyl)amino]propyl}-9H-purin-6-amine

Prepared similarly to Intermediate 14 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate and(tetrahydro-3-furanylmethyl)amine hydrochloride.

LCMS (System D): t_(RET)=2.24 min; MH⁺ 379

Intermediate 17:2-(Butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-9H-purin-6-amine

Prepared similarly to Intermediate 15 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate and2-(tetrahydro-2-furanyl)ethanamine.

LCMS (System C): t_(RET)=0.85 min; MH⁺ 393

Intermediate 18:2-(Butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-2-ylmethyl)amino]propyl}-9H-purin-6-amine

Prepared similarly to Intermediate 14 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate and(tetrahydro-2H-pyran)-2-ylmethyl)amine hydrochloride.

LCMS (System D): t_(RET)=2.55 min; MH⁺ 393

Intermediate 19:2-{3-[6-Amino-2-(butyloxy)-8-(methyloxy)-9H-purin-9-yl]propyl}-1H-isoindole-1,3(2H)-dione

2-(Butyloxy)-8-(methyloxy)-9H-purin-6-amine (7.5 g, 31.6 mmol) wasdissolved in DMF (100 ml) and potassium carbonate (4.37 g, 31.6 mmol)was added. After the suspension had been stirred at room temperature for15 min., 2-(3-bromopropyl)-1H-isoindole-1,3(2H)-dione (8.00 g, 29.8mmol) was added and the suspension was vigorously stirred at roomtemperature for 10 hours. The reaction mixture was extracted with ethylacetate, washed with water, brine and dried. The crude material wasrecrystallized from EtOAc/diethyl ether to give the title compound inthree different batches (2.02 g), (2.44 g) and (1.60 g).

LCMS (System B): t_(RET)=2.16 min; MH⁺ 425

Intermediate 20:9-(3-Aminopropyl)-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine

2-{3-[6-Amino-2-(butyloxy)-8-(methyloxy)-9H-purin-9-yl]propyl}-1H-isoindole-1,3(2H)-dione(6.06 g, 14.28 mmol) was dissolved in ethanol (60 ml) and hydrazinemonohydrate (6.00 ml) was added and stirred at ambient temperature for10 hours. The resultant mixture was concentrated under reduced pressureand the residue suspended in DCM (100 ml) and stirred for 1 hour. Thesuspension was filtered, and the filtered solid washed with DCM (10 ml).The filtrate and washings were washed with water (50 ml) and dried. Theorganic solution was concentrated under reduced pressure to give thetitle compound as an off-white solid (2.74 g).

LCMS (System B): t_(RET)=1.10 min; MH⁺ 295

Intermediate 21:2-(Butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-3-ylmethyl)amino]propyl}-9H-purin-6-amine

Tetrahydro-2H-pyran-3-carbaldehyde (19.39 mg, 0.17 mmol) was added to9-(3-aminopropyl)-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine (50 mg,0.17 mmol) in DCM (1 ml). Sodium triacetoxyborohydride (72.0 mg, 0.34mmol) and DCM (1 ml) was added followed by acetic acid (0.024 ml, 0.425mmol). The mixture was stirred at 20° C. under nitrogen for 5 hours. Themixture was quenched with NaHCO₃ (5 ml) and diluted with DCM (2 ml). Theorganic layer was separated using a hydrophobic frit and the solventremoved in vacuo. The sample was dissolved in 1:1 MeOH:DMSO (1 ml) andpurified by Mass Directed AutoPrep (Method A). The solvent was driedunder a stream of nitrogen to give the title compound as white solid (47mg).

LCMS (System D): t_(RET)=2.40 min; MH⁺ 393

Intermediate 22:2-(Butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-9H-purin-6-amine

Prepared similarly to Intermediate 15 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate and(tetrahydro-2H-pyran-4-ylmethyl)amine.

LCMS (System D): t_(RET)=2.32 min; MH⁺ 393

Intermediate 23:2-(butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-2-ylethyl]amino}propyl)-9H-purin-6-amine

2-(2-Bromoethyl)tetrahydro-2H-pyran (32.8 mg, 0.17 mmol) and9-(3-aminopropyl)-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine (50 mg,0.17 mmol) were dissolved in DMF (2 ml). Triethylamine (0.024 ml, 0.17mmol) was added and the reaction stirred at rt for 16 hours. Additional2-(2-bromoethyl)tetrahydro-2H-pyran (32.8 mg, 0.17 mmol) andtriethylamine (0.024 ml, 0.17 mmol) were added and the mixture washeated at 50° C. for 3 hours. The mixture was diluted with DCM (5 ml)and washed with water (2 ml). The organic layer was separated using ahydrophobic frit and the solvent removed on the nitrogen blowdown unit.The sample was dissolved in 1:1 MeOH:DMSO (1 ml) and purified by MassDirected AutoPrep (Method A). The solvent was dried under a stream ofnitrogen to give the title compound as clear oil (18 mg).

LCMS (System D): t_(RET)=2.55 min; MH⁺ 407

Intermediate 24:2-(Butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-3-ylethyl]amino}propyl)-9H-purin-6-amine

Prepared similarly to Intermediate 23 from9-(3-aminopropyl)-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine and3-(2-bromoethyl)tetrahydro-2H-pyran.

LCMS (System D): t_(RET)=2.42 min; MH⁺ 407

Intermediate 25:2-(Butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-4-ylethyl]amino}propyl)-9H-purin-6-amine

Prepared similarly to Intermediate 15 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1,3-dibromopropane, and 2-(tetrahydro-2H-pyran-4-ylethyl)amine.

LCMS (System D): t_(RET)=2.39 min; MH⁺ 407

Intermediate 26:2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-9H-purin-6-amine

Prepared similarly to Intermediate 15 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate, 1,3-dibromopropane, and(tetrahydro-2-furanylmethyl)amine.

LCMS (System C): t_(RET)=0.98 min; MH⁺ 393

Intermediate 27:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-9H-purin-6-amine

Prepared similarly to Intermediate 15 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate, 1,3-dibromopropane, and[2-(tetrahydro-2-furanyl)ethyl]amine.

LCMS (System C): t_(RET)=0.89 min; MH⁺ 407

Intermediate 28:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-9H-purin-6-amine

Prepared similarly to Intermediate 15 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate, 1,3-dibromopropane, and(tetrahydro-2H-pyran-4-ylmethyl)amine.

LCMS (System C): t_(RET)=0.97 min; MH⁺ 407

Intermediate 29:N²-Butyl-8-(methyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-9H-purine-2,6-diamine

Prepared similarly to Intermediate 15 fromN²-butyl-8-methoxy-9H-purine-2,6-diamine trifluoroacetic acid salt,1,3-dibromopropane, and 2-(tetrahydro-2H-pyran-4-ylethyl)amine.

LCMS (System C): t_(RET)=0.91 min; MH⁺ 378

Intermediate 30:N²-Butyl-8-(methyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-9H-purine-2,6-diamine

Prepared similarly to Intermediate 26 fromN²-butyl-8-methoxy-9H-purine-2,6-diamine trifluoroacetic acid salt,1,3-dibromopropane, and [2-(tetrahydro-2-furanyl)ethyl]amine.

LCMS (System C): t_(RET)=0.9 min; MH⁺ 392

Intermediate 31:N²-Butyl-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-9H-purine-2,6-diamine

Prepared similarly to Intermediate 15 fromN²-butyl-8-methoxy-9H-purine-2,6-diamine trifluoroacetic acid salt,1,3-dibromopropane, and (tetrahydro-2H-pyran-4-ylmethyl)amine.

LCMS (System C): t_(RET)=0.89 min; MH⁺ 392

Intermediate 32:N²-Butyl-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-2-ylethyl]amino}propyl)-9H-purine-2,6-diamine

Prepared similarly to Intermediate 14 fromN²-butyl-8-methoxy-9H-purine-2,6-diamine trifluoroacetic acid salt,1,3-dibromopropane, and 2-(tetrahydro-2H-pyranyl-2-ylethyl)aminehydrochloride.

LCMS (System C): t_(RET)=0.98 min; MH⁺ 406

Intermediate 33: 2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N,O-bis(trimethylsilyl)acetamide (975 ml, 3.988 mol) was added to astirred suspension of 2-fluoro-1H-purin-6-amine (200 g, 1.306 mmol)(available from, for example, AlliedSignal, US) in anhydrousacetonitrile (4 L) in a 10 L controlled lab reactor and the resultingmixture heated to reflux and maintained at that temperature for 2 hours.The circulator was then re-programmed and the reaction mixture cooled to0° C. A solution of tetrahydropyranyl acetate (preparation described inTetrahedron Letters 2006, 47(27), 4741) (282 g, 1.959 mol) in anhydrousacetonitrile (500 ml) was then added slowly via a dropping funnelfollowed by trimethylsilyl trifluoromethanesulfonate (283 ml, 1.567 mol)dropwise via a dropping funnel. No significant exotherm was observed.The circulator temperature was re-adjusted to 10° C. and stirringmaintained for a further 1 hour. The mixture was then quenched byaddition of 1M sodium carbonate (4 L). A solid precipitate was observedand the pH checked to be basic. Additional water was added to thesuspension (1 L) and on standing the layers separated with the aqueouslayer containing significant solid inorganics. The majority of theaqueous and inorganic solid was separated. The organic layer stillcontained significant solid and was cooled to 0° C. with stirring toencourage further precipitation. The solid was the collected byfiltration and the pad was washed very well with water then dried invacuo at 40° C. overnight to give the title compound as a cream colouredsolid (152.8 g).

LCMS (System D): t_(RET)=1.71 min; MH⁺=238

Intermediate 34:9-(4-Chlorobutyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine

2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-amine (1 g, 2.74mmol) and potassium carbonate (0.946 g, 6.84 mmol) in DMF (12.5 ml) werestirred under nitrogen and heated to 50° C. for 1 hour. The mixture wasthen allowed to cool to ambient temperature and 1-bromo-4-chlorobutane(0.315 ml, 2.74 mmol) was added and stirring was continued undernitrogen at ambient temperature overnight. The mixture was thenpartitioned between ethyl acetate and water. The aqueous layer waswashed with ethyl acetate and the combined organic extracts were washedwith saturated brine and passed through a hydrophobic frit andevaporated in vacuo. The residue (1.0322 g) was dissolved indichloromethane and loaded onto an amino cartridge (70 g) and elutedusing a 0-100% ethyl acetate in cyclohexane gradient over 40 minutes.Product fractions were combined and evaporated to give the titlecompound as an off-white solid (667 mg).

LCMS (System D): t_(RET)=3.01 min; MH⁺=342/344

Intermediate 35:9-(5-Chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine

2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-aminetrifluoroacetate (600 mg, 1.642 mmol) and potassium carbonate (567 mg,4.11 mmol) were stirred at 60° C. in DMF (10 ml) for 1 hour undernitrogen. The reaction was cooled to room temperature when1-bromo-5-chloropentane (0.216 ml, 1.642 mmol) and triethylamine (0.343ml, 2.464 mmol) were added and the mixture stirred at 20° C. undernitrogen for 16 hours. The mixture was then diluted with water (10 ml)and brine (10 ml) and extracted with DCM (2×10 ml). The combined organicextracts were evaporated and the residue dissolved in DCM and purifiedby column chromatography using the Flashmaster II (70 g aminopropylcartridge) with a 0-100% ethyl acetate in cyclohexane gradient over 40minutes. The appropriate fractions were combined and evaporated in vacuoto give the title compound as a yellow gum (430 mg).

LCMS (System D): t_(RET)=4.15 min; MH⁺=356/358

Intermediate 36:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-[4-(tetrahydro-2H-pyran-4-ylamino)butyl]-9H-purin-6-amine

9-(4-Chlorobutyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine(70 mg, 0.205 mmol) was mixed with N,N-diisopropylethylamine (0.179 ml,1.024 mmol), sodium iodide (30.7 mg, 0.205 mmol) andtetrahydro-2H-pyran-4-amine (62.1 mg, 0.614 mmol) in acetonitrile (1.5ml) and heated at 70° C. under nitrogen overnight and then left atambient temperature over the weekend. The acetonitrile was evaporatedunder nitrogen and the residue partitioned between aqueous sodiumbicarbonate and dichloromethane. The aqueous phase was re-extracted withdichloromethane and the combined organic extracts were passed through ahydrophobic frit and evaporated under nitrogen to give an amber oil.This material was dissolved in 1:1 DMSO:MeOH (2×1 ml) and purified intwo injections by Mass Directed AutoPrep (Method A). Product containingfractions were combined evaporated under nitrogen in a blowdown unit togive the title compound as a colourless oil (30.35 mg).

LCMS (System D): t_(RET)=2.36 min; MH⁺ 407

Intermediate 37:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-[5-(tetrahydro-2H-pyran-4-ylamino)pentyl]-9H-purin-6-amine

Prepared similarly to Intermediate 36 from9-(5-chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amineand tetrahydro-2H-pyran-4-amine but with heating at 70° C. for anadditional 20 hours.

LCMS (System D): t_(RET)=2.46 min; MH⁺ 421

Intermediate 38:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-{4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]butyl}-9H-purin-6-amine

Prepared similarly to Intermediate 36 from9-(4-chlorobutyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amineand (tetrahydro-2H-pyran-4-ylmethyl)amine.

LCMS (System D): t_(RET)=2.43 min; MH⁺ 421

Intermediate 39:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-{5-[(tetrahydro-2H-pyran-4-ylmethyl)amino]pentyl}-9H-purin-6-amine

Prepared similarly to Intermediate 36 from9-(5-chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amineand (tetrahydro-2H-pyran-4-ylmethyl)amine but with heating at 70° C. foran additional 20 hours.

LCMS (System D): t_(RET)=2.53 min; MH⁺ 435

Intermediate 40:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-{4-[(3R)-tetrahydro-3-furanylamino]butyl}-9H-purin-6-amine

Prepared similarly to Intermediate 36 from9-(4-chlorobutyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amineand (3R)-tetrahydro-3-furanamine but with heating at 70° C. for anadditional 20 hours.

LCMS (System D): t_(RET)=2.32 min; MH⁺ 393

Intermediate 41:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-{4-[(3S)-tetrahydro-3-furanylamino]butyl}-9H-purin-6-amine

Prepared similarly to Intermediate 40 from9-(4-chlorobutyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amineand (3S)-tetrahydro-3-furanamine.

LCMS (System D): t_(RET)=2.33 min; MH⁺ 393

Example 16-Amino-2-(butyloxy)-9-{3-[(2R)-tetrahydro-2-furanylamino]propyl}-7,9-dihydro-8H-purin-8-one

2-(Butyloxy)-8-(methyloxy)-9-{3-[(2R)-tetrahydro-2-furanylamino]propyl}-8,9-dihydro-7H-purin-6-amine(3 mg, 8.23 μmol) was stirred with 4M HCl in 1,4 dioxane (0.05 ml, 0.2mmol) in methanol (2 ml) at 40° C. for 3 hours. The solvent was strippedusing the Radleys blowdown apparatus under a stream of nitrogen. Thesample was loaded in methanol and purified by SPE (2 g) on anaminopropyl (NH₂) cartridge using methanol. The appropriate fractionswere combined and dried under a stream of nitrogen to give the titlecompound as a white solid (2.8 mg).

LCMS (System D): t_(RET)=1.96 min; MH⁺ 351

Example 26-Amino-2-(butyloxy)-9-{3-[(2S)-tetrahydro-2-furanylamino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-{3-[(2S)-tetrahydro-2-furanylamino]propyl}-8,9-dihydro-7H-purin-6-amine.

LCMS (System C): t_(RET)=0.77 min; MH⁺ 351

Example 36-Amino-2-(butyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.13 min; MH⁺ 365

Example 46-Amino-2-(butyloxy)-9-{3-[(tetrahydro-3-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

2-(Butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-3-furanylmethyl)amino]propyl}-9H-purin-6-amine(11 mg, 0.029 mmol) and 4M HCl in 1,4 dioxane (0.160 ml, 0.639 mmol)were stirred at 20° C. for 16 hours in methanol (1 ml). The solvent wasremoved by N₂ blowdown. The sample was loaded in methanol and purifiedby SPE on an aminopropyl (NH₂) cartridge (2 g) using methanol. Theappropriate fractions were combined and dried under a stream of nitrogento give the crude product as a white solid. The sample were dissolved in1:1 MeOH:DMSO (1 ml) and purified by Mass Directed AutoPrep (Method A).The solvent was dried under a stream of nitrogen in the Radleys blowdownapparatus to give the title compound as a white solid (5.6 mg).

LCMS (System D): t_(RET)=2.03 min; MH⁺ 365

Example 56-Amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.16 min; MH⁺ 379

Example 66-Amino-2-(butyloxy)-9-{3-[(tetrahydro-2H-pyran-2-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 4 from2-(butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-2-ylmethyl)amino]propyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.29 min; MH⁺ 379

Example 76-Amino-2-(butyloxy)-9-{3-[(tetrahydro-2H-pyran-3-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-3-ylmethyl)amino]propyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.15 min; MH⁺ 379

Example 86-Amino-2-(butyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.08 min; MH⁺ 379

Example 96-Amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-2-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-2-yl)ethyl]amino}propyl)-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.33 min; MH⁺ 393

Example 106-Amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-3-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-3-yl)ethyl]amino}propyl)-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.19 min; MH⁺ 393

Example 116-Amino-2-(butyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-4-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-4-yl)ethyl]amino}propyl)-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.15 min; MH⁺ 393

Example 126-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 4 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.27 min; MH⁺ 379

Example 136-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 4 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-9H-purin-6-amine.

LCMS (System C): t_(RET)=0.9 min; MH⁺ 393

Example 146-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.22 min; MH⁺ 393

Example 156-Amino-2-(butylamino)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 fromN²-butyl-8-(methyloxy)-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-9H-purine-2,6-diamine.

LCMS (System D): t_(RET)=2.11 min; MH⁺ 364

Example 166-Amino-2-(butylamino)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 fromN²-butyl-8-(methyloxy)-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-9H-purine-2,6-diamine.

LCMS (System D): t_(RET)=2.14 min; MH⁺ 378

Example 176-Amino-2-(butylamino)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 fromN²-butyl-8-(methyloxy)-9-{3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]propyl}-9H-purine-2,6-diamine.

LCMS (System D): t_(RET)=2.06 min; MH⁺ 378

Example 186-Amino-2-(butylamino)-9-(3-{[2-(tetrahydro-2H-pyran-2-yl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 4 fromN²-butyl-8-(methyloxy)-9-(3-{[2-(tetrahydro-2H-pyran-2-yl)ethyl]amino}propyl)-9H-purine-2,6-diamine.

LCMS (System D): t_(RET)=2.33 min; MH⁺ 392

Example 196-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[4-(tetrahydro-2H-pyran-4-ylamino)butyl]-7,9-dihydro-8H-purin-8-one

2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-[4-(tetrahydro-2H-pyran-4-ylamino)butyl]-9H-purin-6-amine(30.35 mg, 0.075 mmol) was dissolved in methanol (2.0 ml) and treatedwith 4M HCl in 1,4 dioxane (0.467 ml, 1.866 mmol) and stirred in acapped vial over the weekend. The solvent was evaporated under nitrogenand the residue was dissolved in methanol and passed down an aminopropylSPE cartridge (2 g, pre-conditioned with 2 column volumes of methanol)and washed through with 1 column volume of methanol. The eluant wasevaporated under nitrogen in a blowdown unit to give the title compoundas an off-white solid (28.34 mg).

LCMS (System D): t_(RET)=2.16 min; MH⁺ 393

Example 206-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(tetrahydro-2H-pyran-4-ylamino)pentyl]-7,9-dihydro-8H-purin-8-one

2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-[5-(tetrahydro-2H-pyran-4-ylamino)pentyl]-9H-purin-6-amine(35.47 mg, 0.084 mmol) was stirred in methanol (2.3 ml) and treated with4M HCl in 1,4 dioxane (0.527 ml, 2.109 mmol). The reaction was stirredin a capped vial overnight and then evaporated under nitrogen and theresidue dissolved in 1:1 DMSO:MeOH (1 ml) and purified by Mass DirectedAutoPrep (Method A). Product-containing fractions were combined andevaporated in a nitrogen blowdown unit to give the title compound as anoff-white gum (23.28 mg).

LCMS (System D): t_(RET)=2.25 min; MH⁺ 407

Example 216-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]butyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 19 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-{4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]butyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.21 min; MH⁺ 407

Example 226-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-{5-[(tetrahydro-2H-pyran-4-ylmethyl)amino]pentyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 20 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-{5-[(tetrahydro-2H-pyran-4-ylmethyl)amino]pentyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.29 min; MH⁺ 421

Example 236-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(3R)-tetrahydro-3-furanylamino]butyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 20 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-{4-[(3R)-tetrahydro-3-furanylamino]butyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.14 min; MH⁺ 379

Example 246-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(3S)-tetrahydro-3-furanylamino]butyl}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 20 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-{4-[(3S)-tetrahydro-3-furanylamino]butyl}-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.14 min; MH⁺ 379

Biological Data

Compounds of the invention were tested for in vitro biological activityin accordance with the following assays, or similar assays:

Assay for the Induction of Interferon-α Using Cryopreserved HumanPeripheral Blood Mononuclear Cells (PBMCs)

Compound Preparation

Compounds were dissolved in DMSO. Serial 2-fold dilutions with DMSO wereprepared and 0.25 μl dispensed into 384-well clear Greiner polypropyleneplates.

Preparation of PBMCs

Blood samples of up to 200 ml were obtained from healthy human donors.Whole blood in 25 ml volumes was overlaid onto 15 ml Ficoll gradients inLeucosep tubes, and centrifuged at 1000 g for 20 min. Cells in the bandat the plasma/histopaque interface were carefully removed and washedtwice with PBS (centrifuged at 400 g for 5 min to harvest). The finalpellet was resuspended in freezing medium (90% Heat-inactivated serum,10% DMSO) to a cell concentration of 4×10⁷ cells/ml. The resuspendedcells were then cryopreserved (frozen) using a rate controlled freezer,and stored at −140° C. for up to 4 months.

Incubation and Assay for Interferon-α

Immediately prior to assay, vials of cryopreserved (frozen) PBMCs werethawed rapidly in a water bath at 37° C. A 1:10 dilution of the cells intrypan blue was prepared and counted. The PBMCs were then diluted ingrowth media [RPMI 1640 containing 10% fetal calf serum (invitrogen),Penicillin+Streptavidin (Gibco cat. #25030-024, 1:50), L-Glutamine 2 mM,and 1000 units/ml recombinant human IFN-gamma (Preprotech catalogue#300-02)] to a density of 1×10⁶ cells/ml, and 50 ul/well dispensed to384-well clear Greiner polypropylene plates containing 0.25 μl DMSO ortest compound in 0.25 μl DMSO. Top final concentration of compound wastypically 50 uM or 5 uM (to obtain curve fit for highly activecompounds). Plates were incubated for 24 h at 37° C. in 5% CO₂.

A multi-isoform immunoassay was used to quantify IFN-α in PBMCsupernatants. Rabbit polyclonal antibody against human IFN-α (cataloguenumber 31101, Stratech Scientific) was diluted 1:10000 in assay buffer(RPMI 1640 containing 10% fetal calf serum, Invitrogen) and 20 μl wasadded to each well of an MSD (Meso-Scale Discovery) single small-spot384-well GAR (goat anti-rabbit antibody coated) plate. The plate wasincubated for 1 h at room temperature with vigorous shaking. Followingthree washes with PBS, 20 μl of cell supernatant were added to each wellof the plate. The plate was then incubated for 1 h at room temperaturewith vigorous shaking. A pair of monoclonal antibodies to IFN-α(catalogue numbers 21100 and 21112, Stratech Scientific) were labelledwith sulfo-TAG (MSD), diluted 1:1000 in assay buffer and 20 μl added toeach well of the plate. The plate was further incubated for 1 h at roomtemperature with vigorous shaking. Following three washes with PBS, 30μl of ×2 T buffer (MSD) was added to each well and the plate was read onan MSD Sector 6000 plate reader.

Data were normalised to internal plate controls of 1 uM resiquimod(n=16) and DMSO (n=16). pEC50 values were derived by 4-parameter curvefit with IRLS in ActivityBase, from 11-point, two-fold serial dilutionof test compounds.

Results

Examples 1 to 24 had a mean pEC₅₀ of >5. Examples 12, 13 and 19 had amean pEC₅₀ of >7.0 and Examples 20 to 24 had a mean pEC₅₀ of >8.0.

Assay for the Induction of Interferon-α and TNF-α Using Fresh HumanPeripheral Blood Mononuclear Cells (PBMCs)

Compound Preparation

Compounds were dissolved and serially diluted in DMSO to give 100× therequired concentration range using a Biomek 2000. 1 ul of test compoundwas transferred into 96-well tissue culture plates using a Biomek FX.Each compound was assayed in duplicate for each donor. Each platecontained a dilution series of the TLR7/8 agonist resiquimod as standardand Column 11 contained 1 μl of 200 μM resiquimod (giving a 2 μM finalconcentration, used to define the approximate maximal response toresiquimod).

Preparation of PBMCs

Blood samples from two human donors were collected into sodium heparin(10 U/ml). 25 ml volumes of whole blood were overlaid onto 15 mlsHistopaque in Leucosep tubes which were centrifuged at 800 g for 20 minand the band at the plasma/histopaque interface carefully removed. Thecollected cells were centrifuged at 2500 rpm for 10 min and the pelletresuspended in 10 ml of media (RPMI 1640 (Low endotoxin) supplementedwith 10% v/v foetal calf serum (FCS, low endotoxin) 100 U/ml penicillinG, 100 μg/ml streptomycin, 10 mM L-glutamine and 1× non-essential aminoacids). A 1:20 dilution of the cells was prepared using trypan blue &the cells counted using a haemocytometer. The PBMCs were diluted to givea final concentration of 2×10⁶/ml and 100 ul of this cells suspensionwas added to wells containing 1 μl of diluted test compound.

Incubation and Assays for Interferon-α and TNF-α

The cell preparations were incubated for 24 hr (37° C., 95% air, 5% CO₂)after which a sample of the supernatant was removed using the Biomek FXand assayed for both IFN-α and TNF-α using the MSD (Mesoscale Discovery)electrochemiluminescence assay platform. The IFN-α assay was carried outsimilarly to that described above. The TNF-α assay was carried out asper kit instructions (Cat No K111BHB).

Cytokine released was expressed as a percentage of the 2 μM resiquimodcontrol (column 11). This percentage was plotted against compoundconcentration and the pEC50 for the response determined by non-linearleast squares curve fitting. For the IFN-α responses generally a 4parameter logistic model was selected. For the TNF responses where aclear maximum response was obtained (i.e. a well defined plateau in theresponse was observed) then a 4 parameter model was generally used. Ifthe upper asymptote of the curve wasn't well defined then the curvefitting was generally constrained to a maximal response of 100% (i.e. tothe response to 2 μM resiquimod) or to the response of the highestconcentration tested if this was greater than the resiquimod response.Some curves were bell shaped for one or both cytokines and the cytokinedata on the down slope of the bell shaped response (i.e. concentrationsabove those giving the maximal response) were generally excluded fromthe fit, usually with the exception of the concentration immediatelyabove the peak response. Curve fitting thus concentrated on the up slopeof the dose response curve.

Results

Examples 3, 5, 8, 9, and 16 showed mean pEC₅₀s for induction of IFN-αand TNF-α of >6 and <5 respectively, and Examples 12, 13, and 14 showedmean pEC₅₀s for induction of IFN-α and TNF-α of >7 and <6 respectively.

The invention claimed is:
 1. A compound of formula (I):

wherein; R¹ is C₁₋₆alkylamino, or C₁₋₆alkoxy; m is an integer having avalue of 3, 4, or 5; n is an integer having a value of 0 to 3; p is aninteger having a value of 1 or 2; or a salt thereof.
 2. A compoundaccording to claim 1, or a salt thereof, wherein R¹ is(1S)-1-methylbutyl]oxy.
 3. A compound according to claim 1, or a saltthereof, wherein n is
 0. 4. A compound according to claim 1, or a saltthereof, wherein n is
 1. 5. A compound according to claim 1, or a saltthereof, wherein n is
 2. 6. A compound or a salt thereof selected fromthe list consisting of:6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{3-[(tetrahydro-2-furanylmethyl)amino]propyl}-7,9-dihydro-8H-purin-8-one;6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-(3-{[2-(tetrahydro-2-furanyl)ethyl]amino}propyl)-7,9-dihydro-8H-purin-8-one;6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(tetrahydro-2H-pyran-4-ylamino)pentyl]-7,9-dihydro-8H-purin-8-one;6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]butyl}-7,9-dihydro-8H-purin-8-one;6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{5-[(tetrahydro-2H-pyran-4-ylmethyl)amino]pentyl}-7,9-dihydro-8H-purin-8-one;6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(3R)-tetrahydro-3-furanylamino]butyl}-7,9-dihydro-8H-purin-8-one,and;6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-{4-[(3S)-tetrahydro-3-furanylamino]butyl}-7,9-dihydro-8H-purin-8-one;and salts thereof.
 7. A pharmaceutical composition comprising a compoundas defined in claim 1, or a pharmaceutically acceptable salt thereof,and one or more pharmaceutically acceptable diluents or carriers.